Anesthetic Effects of Clove Basil Essential Oil (Ocimum gratissimum) Microemulsion on Asian Redtail Catfish (Hemibagrus wyckioides) and Its Biochemical Stress Indicators

Abstract

Ocimum gratissimum (clove basil) essential oil is known for its anesthetic and stress-reducing effects in aquatic animals. In this study, it was synthesized into a microemulsion form and its anesthetic effects on Hemibagrus wyckioides (Asian redtail catfish) juveniles were evaluated. The clove basil essential oil was formulated into a stable oil-in-water microemulsion with a particle size of approximately 36.3 nm and a polydispersity index (PDI) of 0.17. The microemulsion, with concentrations ranging from 125 mg L⁻¹ to 250 mg L⁻¹, effectively induced sedation and anesthesia in fish. It took approximately 2 to 4 min for the fish to reach a state of sedation or anesthesia, depending on the concentration of the clove basil essential oil microemulsion used. After a 30 min induction period using clove basil essential oil microemulsion at concentrations of 125 mg L⁻¹ and 175 mg L⁻¹, the blood cortisol, glucose, and lactate levels, which are stress indicators in fish, were evaluated. The results indicated that the blood cortisol levels in the treatments (6.97 to 7.4 μg dL⁻¹) were consistently lower than in the control group (17.17 μg dL⁻¹) throughout the induction time. However, the glucose (5.6–6.75 mmol L⁻¹) and lactate levels (3.23–5.41 mmol L⁻¹) in the treatment groups increased acutely during the induction time but returned to normal levels (around 3.5 mmol L⁻¹ and 1.6 mmol L⁻¹, respectively) during recovery. This contrasted with the control group, where the lactate and glucose levels remained slightly elevated during the recovery period. Additionally, the recovery time in fish anesthetized with clove basil essential oil microemulsion was consistently short across all of the treatments. These findings highlight the potential application of clove basil essential oil, particularly in microemulsion form, as an effective anesthetic agent for fish.

1. Introduction

In aquaculture, particularly in fish farming, stressors such as transportation, thinning, inspection, and sample collection can weaken the health of aquatic animals, increasing the disease and mortality rate [1]. Anesthesia, whether light sedation or general anesthesia, reduces the sensitivity to external stimuli, lowers activity and stress levels, and decreases the metabolic rate and oxygen demand [2]. These make the fish-handling process easier and less stressful [1,2]. Two types of anesthetics are used in aquaculture: synthetic and natural [2]. Synthetic anesthetics include MS-222 and 2-phenoxyethanol [2]. On the other hand, natural anesthetics are often derived from herbal plants [2]. Herbal plants receive significant attention for future development due to their notable biological activities, such as their anti-stress [3], anti-microbial, and anti-oxidant properties, as well as their ability to enhance immunity [4]. Many plant species from families such as Lamiaceae, Verbenaceae, Lauraceae, and Myrtaceae have been proven to exhibit anesthetic effectiveness [3,4], demonstrating promise for use as an alternative to synthesized anesthetics.

Ocimum gratissimum (clove basil), which belongs to the Lamiaceae family, thrives in tropical climates such as Asia, Africa, and South America [5]. In Vietnam, clove basil is a common herb that grows in plains and low mountains. The essential oil extracted from clove basil contains eugenol as its major active component [6], which is known as a popular anesthesia agent in aquaculture [7]. Besides eugenol, other active components found in clove basil essential oil, such as β-caryophyllene and ocimene [8], also have anesthetic efficacy and stress-reducing properties in aquatic animals. Previous experiments demonstrated the anesthetic effects of clove basil essential oil on various aquatic animals, including silver catfish (Rhamdia quelen) [9] South American catfish (Pseudoplatystoma reticulatum) [10], Nile tilapia (Oreochromis niloticus) [11], and Lophiosilurus alexandri [12]. These effects are evidenced by the essential oil concentrations capable of anesthetizing fish and by changes in biochemical blood parameters, such as cortisol and glucose, which serve as stress indicators in fish [13]. At certain suitable concentrations, clove basil essential oil can effectively anesthetize fish and prevent increases in blood glucose and cortisol levels [10,12].

However, clove basil essential oil also shares some drawbacks with other essential oils, such as insolubility and high volatility. These properties can lead to changes in the biological activity of the essential oils. In this context, encapsulation presents a promising technology to address these issues [14]. Among encapsulation techniques, microemulsions and nanoemulsions have been widely explored, offering numerous benefits [14]. These include enhancing water solubility and protecting essential oils from decomposition by creating physical barriers that protect the active substances from environmental exposure and degradation. This results in the improved stability of the biological activities of essential oils [14,15]. Furthermore, precise distribution allows for a reduction in the required dosage while still ensuring the preservation of biological functionality [16]. In aquaculture, numerous experiments have previously been conducted to prepare micro- or nanoemulsions based on essential oils. These experiments aimed to control bacterial growth in aquaculture environments, limit microbial pathogens [17], and reduce stress in aquatic animals [18], and they have recorded positive results.

In this experiment, clove basil essential oil was synthesized into a microemulsion to improve its solubility and stabilize its biological activities during storage. Then, the anesthetic efficacy of the clove basil microemulsion was assessed in Asian redtail catfish (Hemibagrus wyckioides), a species of high economic value. Additionally, cortisol, glucose, and lactate levels in the fish’s blood were analyzed after induction and recovery time to evaluate the effects of the anesthetic agent on stress indicators in fish blood. The results provide a basis for developing a natural anesthetic for use in aquaculture, aligning with the trend of sustainable aquaculture development.

2. Materials and Methods

2.1. Preparing a Microemulsion Based on Clove Basil Essential Oil

Polysorbate 20 and propylene glycol from St. Louis, USA were used as a surfactant and co-surfactant in this trial. The clove basil essential oil was purchased from the local distillation facility and analyzed using gas chromatography and mass spectrometry (GC–MS) to determine its chemical composition prior to preparing the microemulsion. The analysis was performed using GC–MS equipment (Agilent Technologies, Santa Clara, CA, USA), featuring a stationary phase of 5% diphenyl 95% dimethyl polysiloxane and helium gas as the mobile phase. Distilled water was used as the aqueous phase in the prepared microemulsion.

The appropriate microemulsion formulation was selected from the pseudo-ternary phase diagram, which was constructed using the titration method with water at room temperature. The tested ratios of surfactant to co-surfactant were 1:1, 2:1, and 3:1. Additionally, the essential oil to surfactant mixture ratios evaluated were 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1. Initially, polysorbate 20 and essential oil were mixed using a magnetic stirrer at 700 rpm at room temperature for 30 min. Then, a mixture of water and propylene glycol was added drop by drop in proportions ranging from 10% to 90%. The formation of microemulsion was determined when a clear and homogeneous solution appeared. Several characteristics of the synthesized clove basil essential oil microemulsions were evaluated.

The particle size was measured using a dynamic light scattering device (Horiba LB550, Kyoto, Japan), and the polydispersity index (PDI) was calculated by the following formula [19,20]:

$${\mathrm{P}\mathrm{D}\mathrm{I}=\left({\displaystyle \frac{\mathrm{S}\mathrm{D}}{\mathrm{M}\mathrm{e}\mathrm{a}\mathrm{n} \mathrm{p}\mathrm{a}\mathrm{r}\mathrm{t}\mathrm{i}\mathrm{c}\mathrm{l}\mathrm{e} \mathrm{s}\mathrm{i}\mathrm{z}\mathrm{e}}}\right)}^2$$

where SD: standard deviation.

The electrical conductivity and zeta potential were measured using a Hemera analyzer (Hemera, Grenoble, France) and a Malvern zetasizer instrument (Mavern Panalytical, Malvern, UK), respectively. The viscosity was determined by an mVROC viscometer (RheoSense, San Ramon, CA, USA). The turbidity of the microemulsions was determined at a wavelength of 502 nm using a Cary 100 UV-Vis spectrophotometer (Varian, Palo Alto, CA, USA) and calculated by the following formula [21]:

$$\mathrm{T}\mathrm{u}\mathrm{r}\mathrm{b}\mathrm{i}\mathrm{d}\mathrm{i}\mathrm{t}\mathrm{y}\left(\%\right)={\displaystyle \frac{2.303\times \mathrm{a}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}}{\mathrm{c}\mathrm{u}\mathrm{v}\mathrm{e}\mathrm{t}\mathrm{t}\mathrm{e} \mathrm{w}\mathrm{i}\mathrm{d}\mathrm{t}\mathrm{h}\left(\mathrm{c}\mathrm{m}\right)}}$$

Based on the characteristics of the synthesized microemulsions, a suitable formula was selected for subsequent experiments. Additionally, the stability of the microemulsion was tested through centrifugation at 1604.8× g for 15 min, after which the uniformity of the solution and the change of particle size and PDI were observed. Furthermore, the microemulsion underwent a heating–cooling test to evaluate temperature stability. The solution was placed in a transparent glass storage tube and subjected to alternating storage conditions at 40 °C and 4 °C for six cycles with a cycle of 24 h [22].

2.2. Assessing the Anesthetic Efficacy of Clove Basil Essential Oil Microemulsion in Asian Redtail Catfish

Asian redtail catfish juveniles were transported from a local fish hatchery to our laboratory two weeks before the trials. Due to the short transportation duration (approximately 30 min), the fish were placed in sealed bags with oxygen, and the water was cooled to reduce their activity and minimize stress. The fish were acclimated to the experimental environment by being reared in a 500 L tank with a continuous aerator. Water parameters were carefully controlled to remain within the optimal range for fish (the temperature was 28 °C and the pH was 6.5–6.9). The water was changed twice a week with leftover feed and feces siphoned out during the process. The fish were fed commercial floating pellets containing 30% protein twice daily, with the feed quantity adjusted to ensure satiety.

The anesthetic efficacy of clove basil essential oil microemulsion was tested in Asian redtail catfish juveniles, with an average weight of 15.51 ± 2.90 g and an average length of 12.91 ± 0.93 cm. A total of 120 fish were used in this trial. The fish were exposed to the microemulsion at concentrations of 100, 125, 150, 175, 200, 225, and 250 mg L⁻¹, and a control group (without an anesthetic agent), and the trial was conducted in triplicate. The microemulsion was dispersed into 20 L of water in the tanks, each tank contained 5 fish, and each fish was used only once during the experiment. The stages of anesthesia in fish were monitored and categorized as follows: stage 1: the fish exhibit unusual swimming behaviors or a slight reduction in their response to external stimuli; stage 2: fish become less active and unresponsive to external stimuli; stage 3, further divided into stage 3a and 3b: at stage 3a, fish lose partial equilibrium, and at stage 3b, they completely lose equilibrium; stage 4: fish show no reaction to handling, even when the tail peduncle is impacted [9,23]. The maximum observation time was 10 min, after which the fish were transferred to clean water tanks without the anesthetic agent. Fish were considered recovered when they resumed normal swimming behavior and responded to external stimuli [9,23]. After recovery, the fish were moved to separate 45 L tanks with aerators, where they were monitored for mortality over the following week. Induction and recovery times were measured in seconds using an automatic stopwatch. A linear regression analysis was performed to determine the relationship between recovery time (y) and anesthetic concentrations (x). The coefficient of determination R² was calculated to assess the goodness of fit of the model to experimental data. A high R² value indicates that the regression model is well suited to the observed data.

2.3. Evaluating Various Biochemical Blood Parameters as Stress Indicators in Fish Anesthetized with Clove Basil Essential Oil Microemulsion

Based on the results from the aforementioned anesthetic efficacy of clove basil essential oil microemulsion, its concentrations were chosen for this experiment, including 125 and 175 mg L⁻¹. The experiment involved 3 groups: a control group (without the anesthetic agent), and two groups treated with the specified concentrations of clove basil essential oil microemulsion. The trial was conducted with three replicates, using a total of 81 fish, with 9 fish per tank. The stages of anesthesia were monitored as previously described. The fish were observed for a maximum of 30 min or until they reached stage 4, after which they were transferred to the anesthetic-free water to monitor the recovery process. Blood samples were collected immediately after the induction time and 2 h after the recovery process began. The blood was drawn using a 1 mL sterilized syringe from the caudal peduncle arteries and stored in microtubes containing EDTA for cortisol and glucose analysis, and EDTA–sodium fluoride tubes for the lactate analyzer. Blood samples were kept at 4 °C and analyzed for biochemical parameters on the same day. Blood samples were centrifuged at 7000× g for 15 min to separate the plasma [24]. Cortisol levels were measured using the electro chemiluminescence immunoassay method on the Roche Cobas e801 system (Roche Diagnostics, Rotkreuz, Switzerland). Lactate levels were determined using the lactate oxidase method on the Abbott Alinity analyzer. Glucose levels were quantified by the hexokinase method using the AU5822 chemistry analyzer (Beckman Coulter, Brea, CA, USA). The induction and recovery times at different clove basil essential oil microemulsion concentrations and its effects on fish blood parameters (glucose, cortisol, and lactate levels) in the experimental treatments were evaluated using one-way analysis of variance (ANOVA), followed by Tukey’s test for pairwise comparisons between treatments. Statistical significance was determined at a confidence level of 95%.

3. Results

3.1. Results of Synthesizing Clove Basil Essential Oil Microemulsion

Microemulsion formation was observed through the phase diagrams. Polysorbate 20 and propylene glycol were used in this trial at coordinating ratios of 1:1, 2:1, and 3:1. The 1:1 ratio yielded the smallest microemulsion formation area, while the 3:1 ratio produced the largest microemulsions. With a polysorbate 20 to propylene glycol ratio of 3:1, the results showed that microemulsions were successfully prepared at an essential-oil-to-surfactant mixture ratio of 1:9 at varying water ratios (ranging from 10% to 90%). At an essential-oil-to-surfactant ratio of 2:8, microemulsions were formed at water ratios of less than 60%, while no microemulsion formation occurred at water ratios above 70. Additionally, at a 3:7 ratio, no microemulsion was formed when the water content exceeded 40% (w/w), and the ability to form microemulsions became more limited at higher oil-to-surfactant ratios.

The trial aimed to synthesize an oil-in-water microemulsion that enhances the dispersion of the essential oil and maintains its bioactivity during storage. Therefore, the selected formulations must yield microemulsions with high conductivity, small particle size, and low particle size dispersity. Based on the recorded results, several microemulsion formulations were chosen, and their characteristics are presented in

Table 1. The selected oil-in-water Ocimum gratissimum essential oil microemulsion formulations based on the phase diagram.

Name of Samples	Essential Oil (% w/w)	Water (% w/w)	Ratio of Polysorbate 20 and Propylene Glycol
M1	8	60	2:1
M2	10	50	3:1
M3	8	60	3:1

and

Table 2. The properties of the selected oil-in-water Ocimum gratissimum essential oil microemulsion formulations.

Name of Samples	Particle Size (nm)	PDI	Conductivity (μS/cm)	Viscosity (mPa.s)	Turbidity (%)
M1	44.30 ± 3.44	0.49 ± 0.27	110.50 ± 0.26	66.09 ± 11.18	0.07 ± 0.00
M2	61.37 ± 8.86	1.02 ± 0.24	106.53 ± 0.21	98.95 ± 1.55	0.06 ± 0.01
M3	36.33 ± 1.84	0.17 ± 0.04	124.97 ± 0.42	85.01 ± 11.57	0.07 ± 0.00

(Note: mean ± standard deviation, PDI = (SD/mean particle size)², SD and mean particle size were determined by the dynamic light scattering (DLS) equipment.

.

The microemulsions synthesized using the three selected formulations were pale yellow in color, homogeneous, and transparent. Their particle sizes ranged from 36.33 to 61.37 nm, with PDI values between 0.17 and 1.02. The results confirmed that all three microemulsions (M1-M3) were the oil-in-water type, as indicated by their high conductivity (106.53–124.97 μS cm⁻¹). Additionally, their low turbidity (0.06–0.07%) demonstrated transparency and uniformity. Among the formulations, the M3 microemulsion exhibited the smallest particle size (approximately 36 nm) and the lowest PDI (0.17) (Figure 1), suggesting a narrow particle size distribution, homogeneity, and high stability.

In contrast, the M2 microemulsion exhibited a larger average particle size of approximately 61 nm and a higher PDI value of 1.02, reflecting the instability of the microemulsion. Additionally, the viscosity of all microemulsions ranged from 66.09 to 85.01 mPa.s, which was relatively low, making them more convenient for use. Based on the collected data regarding the characteristics of the three prepared microemulsion formulas, M3 was selected for the stability test and the subsequent trials in fish.

The zeta potential of the selected microemulsion (M3) was −10.28 ± 0.78 mV (Figure 2), which fell within the range of ±30 mV. Despite this, it remained relatively favorable compared with the microemulsions formulated with nonionic surfactants and contributed to the stability of the microemulsion in this trial. Additionally, the stability of the selected microemulsion was evaluated using both centrifugation and thermal cycling tests. After the centrifugation test, the microemulsion remained homogeneous, transparent, and free from phase separation. There were no significant changes in particle size or PDI compared to the pre-centrifugation measurement. Additionally, during the thermal cycling test at 4 °C and 40 °C, the microemulsion was still stable with the particle size remaining consistent and the PDI value increasing slightly but the difference was not significant, as shown in Figure 3.

3.2. Results of Evaluating the Anesthetic Effectiveness of Clove Basil Essential Oil Microemulsion in Asian Redtail Catfish

The results of the anesthetic efficacy of clove basil essential oil microemulsion in Asian redtail catfish juveniles are shown in

Table 3. Induction and recovery time of fish (Hemibagrus wyckioides) exposed to the Ocimum gratissimum essential oil microemulsion at different concentrations.

Stages (Seconds)	Concentrations of Microemulsion (mg L−1)						p-Value
	125	150	175	200	225	250
1	230.33 ± 53.90 a	126.33 ± 10.79 b	115.00 ± 15.59 b	130.33 ± 13.28 b	131.00 ± 9.54 b	121.33 ± 7.51 b	0.001
2	369.67 ± 53.90 a	363.67 ± 13.20 a	144.00 ± 31.18 d b	189.67 ± 44.02 b	156.00 ± 22.27 b	111.00 ± 10.58 b	<0.001
3a	-	110.00 ± 3.46 c	341.00 ± 15.59 a	280.00 ± 33.42 a b	241.00 ± 25.24 b	151.33 ± 41.86 c	<0.001
3b	-	-	-	-	72.00 ± 8.72 b	216.33 ± 44.81 a	0.005
Recovery	63.33 ± 10.41 c	86.00 ± 8.66 b c	125.00 ± 18.03 b c	147.00 ± 15.72 a b	153.00 ± 7.94 a b	212.67 ± 68.13 a	0.001

(Note: Values are presented as mean ± standard deviation (n = 3). Fish were exposed to clove basil essential oil microemulsion (ME) for 10 min before being transferred to clean water tanks for recovery observation. The duration of each anesthesia stage at different ME concentrations was compared. Different superscript letters within the same row indicate a significant difference (p < 0.05, one-way ANOVA with Tukey’s test.

.

In this experiment, clove basil essential oil microemulsion at concentrations of 100–250 mg L⁻¹ was tested on Asian redtail catfish juveniles. The results indicated that at the concentration of 125 mg L⁻¹, the fish were tranquilized, exhibiting reduced activity and no response to external stimuli for 369.67 ± 53.90 s. At concentrations of 150 mg L⁻¹ and higher, the fish experienced partial loss equilibrium (stage 3a), while at 225 mg L⁻¹ and 250 mg L⁻¹, they completely lost equilibrium (stage 3b) within 72.00 ± 8.72–216.33 ± 44.81 s. However, there was no significant correlation between the induction time at each stage and the increasing microemulsion concentrations. In contrast, the recovery time of the fish at all tested concentrations of clove basil essential oil microemulsion was less than 4 min and showed a positive correlation with the microemulsion concentrations, with an R² value of approximately 0.95 (Figure 4).

In the control group (without an anesthetic), the fish remained active without showing any abnormal activity or mucus loss. Furthermore, all fish exposed to clove basil essential oil microemulsion at various concentrations survived, with no abnormalities or mortality recorded during the trial and the subsequent one-week observation period. The primary component of clove basil (Ocimum gratissimum) essential oil in this study, determined by GC–MS analysis, was eugenol (85.4%), along with several other compositions in lower proportions, including β-ocimene (5.2%), β-cubebene (4.4%), β-caryophyllene (1.8%), σ-cadinene (0.9%), and other minor compositions. The composition of clove basil essential oil helps supports the anesthetic efficacy of its microemulsion, as it contained components with known anesthetic properties for fish, such as eugenol, caryophyllene, and ocimene.

3.3. Results of Glucose, Cortisol, and Lactate Levels of Fish Anesthetized with Clove Basil Essential Oil Microemulsion

Fish are anesthetized to varying degrees based on purpose and duration. During transportation, light anesthesia is typically used to reduce metabolic activity, oxygen consumption, CO₂ and NH₃ production, and physiological stress [25,26], which lowers mortality rates. In contrast, deep anesthesia minimizes all physical activities, including operculum movements, which becomes a stress factor as fish cannot obtain oxygen, leading to increased CO₂ concentration in their blood [27]. This leads to elevated plasma cortisol and adrenaline levels, and without proper gill flushing it can result in mortality [27]. Therefore, in this trial, two concentrations of microemulsion were selected to ensure fish safety while inducing different anesthesia levels: 125 mg L⁻¹ for sedation and 175 mg L⁻¹ for partial anesthesia (stage 3a). These levels are commonly used in practices such as transportation, pond transfer, or experimental handling. Fish anesthetized with the clove basil microemulsion were evaluated for blood biochemical parameters as stress indicators.

Throughout the 30 min observation, fish exposed to clove basil essential oil microemulsion at 125 mg L⁻¹ were anesthetized up to stage 2 and their recovery time after being transferred to clean water was approximately 122.67 ± 29.69 s, while fish anesthetized with the 175 mg L⁻¹ concentration reached stage 3. During this stage, fish were inactive and partially lost equilibrium for around 1434.0 ± 103.3 s before being transferred to clean water. The recovery time for fish in these treatments was 268.00 ± 49.00 s. Blood samples were collected at two time points: after the induction period and after a two-hour recovery period to analyze stress-specific indicators, including cortisol, glucose, and lactate levels. The water quality parameters in the experimental tanks were maintained within the optimal range for the fish as follows: pH 6.5–6.9, temperature 26–27 °C, dissolved oxygen levels 4.5–5.5 mg L⁻¹, and ammonia levels near zero. This ensured that any effects observed in the fish were attributable solely to the anesthetic agent and not influenced by water quality conditions.

After the induction period, the cortisol levels in fish treated with clove basil essential oil microemulsion as an anesthetic ranged from 6.97 to 7.4 μg dL⁻¹, significantly lower than in the control treatment (17.17 μg dL⁻¹) (p = 0.006) (Figure 5). Following a recovery in clean water, fish in the 125 mg L⁻¹ treatment resumed normal activity after approximately 2 min, and those in the 175 mg L⁻¹ treatment took around 4–5 min. After 2 h of recovery, the cortisol levels in the 125 mg L⁻¹ and 175 mg L⁻¹ treatments were 12.14 μg dL⁻¹ and 7.15 μg dL⁻¹, respectively, both remaining lower than in the control group at the same time. However, in the 125 mg L⁻¹ treatment, the cortisol level showed no significant difference compared to the control group and exhibited a slight increase relative to the levels immediately after induction. In contrast, the cortisol levels in the 175 mg L⁻¹ treatment remained unchanged from post-induction and were significantly lower than those in the control group (p = 0.035) (Figure 5).

In addition to cortisol, two secondary stress-related indicators, lactate and glucose, were also recorded (Figure 6 and Figure 7). Following the induction period of 30 min, in the 125 mg L⁻¹ and 175 mg L⁻¹ treatments, the glucose levels achieved 5.60 ±1.23 mmol L⁻¹ and 6.75 ± 0.93 mmol L⁻¹, respectively, but the highest glucose levels were recorded in the control group at 8.08 ± 2.28 mmol L⁻¹. However, these differences were not statistically significant (p = 0.241) between the experimental and control groups. After two hours of recovery in clean water, the blood glucose levels in the 125 mg L⁻¹ and 175 mg L⁻¹ treatments decreased compared with the end of the induction period and were significantly lower than those in the control group (p = 0.019) (Figure 6).

After the 30 min induction period, lactate levels increased slightly across all trial groups, with the highest concentration observed in the 175 mg L⁻¹ treatment at 5.41 ± 0.66 mmol L⁻¹. This value was significantly higher than those in the other two groups (p = 0.026), while the lactate levels in the 125 mg L⁻¹ treatment and the control group, both around 3.2 mmol L⁻¹, showed no significant difference. After 2 h of recovery in clean water, the lactate levels in the control group slightly decreased but remained higher than those in the other treatments at approximately 2.35 ± 1.04 mmol L⁻¹. In contrast, the lactate concentrations in the 125 mg L⁻¹ and 175 mg L⁻¹ groups significantly dropped compared with their post-induction levels, reaching approximately 1.63 ± 0.87 mmol L⁻¹ and 1.57 ± 0.19 mmol L⁻¹, respectively. However, no significant differences were observed among all experimental treatments at this stage (p = 0.502) (Figure 7).

4. Discussion

4.1. Characteristics of the Synthesized Clove Basil Essential Oil Microemulsion

The PDI (polydispersity index), which typically ranges from 0 to 1, characterizes the particle size distribution within the solution [19]. A PDI closer to 0 indicates a more homogeneous (mono-disperse) solution [19]. Studies have suggested that a PDI of less than 0.3 signifies a homogeneous solution with a narrow particle size distribution [28,29,30], whereas a PDI greater than 0.7 reflects a wide particle size distribution range [30]. In our study, only the M3 microemulsion had a PDI value below 0.3, indicating its high uniformity.

Additionally, electrical conductivity is a key parameter for determining the type of microemulsion. Typically, the electrical conductivity of an oil-in-water microemulsion is higher than that of a water-in-oil microemulsion [31]. In our trial, the electrical conductivity of the three microemulsion formulations increased with rising water content. In particular, M1 and M3, which contained 60% water, exhibited higher electrical conductivity compared with the remaining formulation. This finding aligns with the results reported by R. Sha et al. (2017), where the electrical conductivity of a microemulsion increased from 10 to 150 μS cm⁻¹ as the water content rose [32]. When the water content exceeded 50.2%, the system transitioned to an oil-in-water microemulsion, resulting in an electrical conductivity of 140–150 μS cm⁻¹ [32]. These results suggest that all three experimental microemulsions were oil-in-water microemulsions.

The viscosity of the prepared microemulsions ranged from 66.09 to 85.01 mPa.s, with a slight increase corresponding to higher surfactant content. The viscosity of a microemulsion can be influenced by several factors, including the viscosity of the dispersed and continuous phase, the types and concentrations of surfactant and co-surfactant, as well as the droplet sizes in solutions [33]. In our study, the viscosity of the microemulsions may be due to the use of polysorbate 20, which has a high viscosity. However, the high viscosity also contributed to the stability of the microemulsions, which is related to the diffusion constant and the interactions between droplets [34]. Higher viscosity results in a lower diffusion constant, reducing the frequency of droplet collisions. This reduction in collisions helps prevent phase separation, thereby enhancing the stability of the microemulsion system [34]. This was related to the stability of the M3 microemulsion formulation, which has a viscosity of 85.01 mPa.s.

The zeta potential value represents the surface charge of particles within a colloidal system, influencing the stability of particle distribution systems such as micro- or nanoemulsions. A high zeta potential enhances the repulsive forces among particles, preventing aggregation, thereby increasing the stability of the system [35]. For a microemulsion utilizing ionic surfactants, the zeta potential can exceed +30 mV and −30 mV [36], indicating a stable microemulsion. However, microemulsions containing nonionic surfactants typically exhibit lower zeta potentials, and this value can be closer to 0 [36]. Despite this, stability has also been observed in microemulsions with low zeta potential in some studies, suggesting that it is not the sole mechanism influencing microemulsion stability.

Previous experiments have also reported similar results regarding low zeta potential in stable colloidal systems. For instance, in a study developing a microemulsion to enhance the solubility of flunarizine using Tween 80, Capmul CMC and PEG400 as the surfactant and co-surfactant, the zeta potential was −6.34 mV, and the system remained stable for six months [37]. Another study, which synthesized Syzygium aromaticum essential oil microemulsion with the surfactant and co-surfactant of Tween 80 and PEG 400, found a zeta potential of −5.36 mV. However, the microemulsions remained stable after undergoing centrifugation and heating–cooling cycling tests [38]. In our study, the selected microemulsion had a zeta value of −10.28 ± 0.78 mV, within the ±30 mV range. This low zeta potential was due to the use of polysorbate 20, a nonionic surfactant. However, compared with previous trials with nonionic surfactants, this value was relatively high. This contributed to the stability of the microemulsion, as shown by its homogeneity during centrifugation and the thermal cycling test, with no signs of increased particle size or phase separation.

4.2. Anesthetic Efficacy of Clove Basil Essential Oil Microemulsion

The anesthetic efficacy of clove basil essential oil has also been demonstrated in various catfish species. For instance, silver catfish Rhamdia quelen with an average weight of 9.0 ± 0.2 g and an average length of 10.3 ± 0.1 cm were anesthetized by clove basil oil at a concentration of 10 mg L⁻¹, and they reached stage 4 at 30 mg L⁻¹. Notably, no mortality or adverse effects were observed in fish exposed to concentrations ranging from 30 to 300 mg L⁻¹ [9]. However, the recovery time ranged from 7 to 14 min at anesthetic concentrations of 30 and 70 mg L⁻¹ [9], which was notably longer than in our trial. This could be attributed to the higher concentrations of essential oil used and the difference in the fish size. However, another study demonstrated that fish anesthetized with an essential oil microemulsion had a shorter recovery time compared with those anesthetized with original essential oil [39]. This experiment also indicated that the effectiveness of a cinnamon essential oil nanoemulsion was higher than original cinnamon essential oil at stage 3b at the similar concentration of 100 mg L⁻¹ [39]. This can also explain why the recovery time for fish was relatively short (less than 5 min) when using clove basil essential oil microemulsion at the different concentrations in our trial.

In another study conducted on South American catfish (Pseudoplatystoma reticulatum) with an average weight of 40.0 ± 9.9 g, clove basil essential oil at an optimum concentration of 187.81 mg L⁻¹ induced anesthesia with an induction time of 222.02 s [10]. The recovery time ranged from 197.8 to 276.2 s, which positively correlated with experimental concentrations between 100 and 200 mg L⁻¹ [10]. However, the major anesthetic component of clove basil essential oil in this experiment, eugenol, only accounted for 39.5% [10]. Compared with our trial, both the induction and recovery times were shorter. This difference was due to the size of fish and the eugenol content in the clove basil essential oil used.

In our study, eugenol was the major component of clove basil essential oil, comprising 85.4% of the essential oil, and it was primarily responsible for the oil’s anesthetic efficacy. Other components presented in smaller amounts that also contributed to anesthetic activity included β-caryophyllene (1.8%) and β-ocimene (5.2%). Eugenol has long been recognized as an effective anesthetic in various fish species [7,40,41], including catfish [42]. Moreover, β-caryophyllene has been reported to possess anesthetic properties in fish as observed when presented in some essential oils having anesthetic activity [43]. Similarly, ocimene, a component of clove basil essential oil, has been reported to exhibit anesthetic properties in aquatic animals [8]. The ratio of these components in the essential oil can significantly impact its anesthetic activity, as well as determine the effective concentrations required for anesthesia in fish. These findings help to explain the anesthetic efficacy of clove basil essential oil microemulsion on Asian redtail catfish and the observed difference in the induction and recovery times of fish in our trial compared with previous studies.

4.3. Changes of the Blood Cortisol, Lactate, and Glucose Levels in Fish Anesthetized with Clove Basil Essential Oil Microemulsion

Several previous studies have demonstrated that cortisol is a specific indicator of stress in fish. In Asian redtail catfish (Hemibagrus nemurus), plasma cortisol levels increased when exposed to a change of water salinity for 12 days, with levels ranging from around 20 to 90 ng mL⁻¹ (2–9 μg dL⁻¹) [44]. Similarly, a study on three spot gourami (Trichogaster trichopterus) indicated that acute stress, caused by net capturing and transferring out of tanks, led to an increase in cortisol levels to 190 ng mL⁻¹ two hours after handling. In contrast, fish anesthetized by tricaine methanesulfonate exhibited a cortisol level of 60 ng mL⁻¹, significantly lower than in the control group [45]. Cortisol levels in fish blood after stress can range from 40 to 200 ng mL⁻¹, depending on fish species, and may even reach up to 1000 ng L⁻¹ (equivalent to 100 μg dL⁻¹) [45].

In our study, the results showed that clove basil essential oil microemulsion at concentrations of 125 mg L⁻¹ and 175 mg L⁻¹ effectively reduced stress in fish after 30 min exposure time, as evidenced by the cortisol levels in two treatments (6.97 to 7.4 μg dL⁻¹) which were significantly lower than in the control group (17.17 μg dL⁻¹) (p < 0.05). However, after two hours of recovery, the cortisol levels in the 125 mg L⁻¹ treatment increased, though they remained lower than those of the control group. This increase could be attributed to the fact that fish in the 125 mg L⁻¹ treatment only reached stage 2 anesthesia and were subjected to net capture and transfer to clean water during the recovery stage, which likely acted as stressors, contributing to the change in cortisol levels. In contrast, the cortisol levels of fish in the 175 mg mL⁻¹ treatment remained significantly lower than those in the control group and did not differ from the levels observed immediately post-induction.

N. A. Pamukas et al. (2021) reported that normal blood glucose levels in Asian redtail catfish (Hemibagrus nemurus) typically ranged between 56 and 84.33 mg dL⁻¹ (equivalent to 3.11–4.86 mmol L⁻¹) [46]. When fish were exposed to stressors, their blood glucose levels increased to control energy utilization [46]. This rise in glucose results from the release of stress-induced hormones, primarily epinephrine and norepinephrine, which stimulate glycogenolysis in the muscle and liver, releasing glucose to meet elevated energy demands during and after stress [47]. Additionally, previous studies have also suggested that anesthesia can induce hyperglycemia in fish [48,49]. L. Silva et al. (2013) reported similar findings when evaluating blood parameters in catfish (Rhamdia quelen) anesthetized by Hesperozygis ringens and Ocotea acutifoliab essential oils [48]. After 7 min of exposure to Hesperozygis ringens essential oil, blood glucose levels were elevated compared with the control group, though the difference was not statistically significant. However, fish exposed to Ocotea acutifolia essential oil exhibited significantly higher glucose levels compared with the control group [48].

In our trial, an increase in glucose levels was recorded across all trial groups after the 30 min induction time. This rise could contribute to stressors caused by handling and transferring the fish to a new environment as well as the use of clove basil essential oil microemulsion as an anesthetic. However, the elevated glucose levels in the treatments were acute and returned to normal values after recovery in clean water. In contrast, the blood glucose level in the control group remained elevated even after a 2 h recovery period in clean water.

Similar to glucose, lactate levels in fish blood could be influenced by stressors and other factors. For example, the lactate level in the blood of Sparus aurata reached 7.51 mmol L⁻¹ when subjected to acute handling stress three times over 30 min, and the glucose levels also rose to 254.00 mg dL⁻¹ [47]. However, in another experiment by C. S. Hanley et al. (2010), the blood lactate concentrations in three species of perch, koi, and walleye ranged from 1.21 mmol L⁻¹ to 7.81 mmol L⁻¹ [50]. During anesthesia with tricaine methanesulfonate, lactate levels increased from an initial range of 1.2–2 mmol L⁻¹ to 5.33–7.81 mmol L⁻¹ by the end of the anesthesia process, a level sufficient for surgical procedures [50]. Blood lactate is a byproduct of the anaerobic metabolism process, and can be affected by anesthesia. Anesthesia can impair the respiratory capacity of fish, causing muscles to change towards the anaerobic metabolism, thereby leading to a lactate increase in fish blood [47,49]. Moreover, others factors such as water temperature or the time of day also contribute to elevate lactate levels in fish blood [50].

These findings align with our experimental results, where the highest lactate level was observed in the 175 mg L⁻¹ treatment, corresponding to fish being anesthetized at stage 3a. Subsequently, it decreased to the normal level during the recovery time. These results suggest that the stress resulting from capture, handling, or environmental transfer may persist for an extended duration. The use of clove basil essential oil microemulsion as an anesthetic appears to mitigate this stress, facilitating a quicker return to normal physical condition in the fish. However, the use of clove basil essential oil microemulsion can increase acute glucose and lactate levels in anesthetized fish.

5. Conclusions

Clove basil essential oil was formulated into a stable oil-in-water microemulsion with a particle size of 36 nm and a PDI of 0.17. This microemulsion effectively anesthetized Asian redtail catfish juveniles at concentrations ranging from 125 mg L⁻¹ to 250 mg L⁻¹, with consistently short recovery times across treatments. At a concentration of 225 mg L⁻¹ and above, the fish achieve deep anesthesia, characterized by complete loss of equilibrium. Several blood stress indicators in fish demonstrated that clove basil essential oil microemulsion effectively reduced stress responses, as evidenced by the lower cortisol level during the induction period. Although it caused an acute increase in blood glucose and lactate levels, these values normalized during recovery. These findings suggest the potential for developing clove basil essential oil microemulsion as an anesthetic agent in aquaculture. However, further studies are necessary to assess the impact of surfactants and the higher concentrations of clove basil essential oil microemulsion on fish, as well as to determine optimal methods for different applications in aquaculture.