Short-Term Anesthesia with Clove Oil and Propofol: Physiological Responses in Persian Sturgeon (Acipenser persicus)

Abstract

Anesthesia is commonly used in sturgeon aquaculture to reduce stress and pain during handling and procedures. This study compared the effects of clove oil (rich in eugenol) and propofol—an anesthetic widely used in human and veterinary medicine—on Persian sturgeon (Acipenser persicus) fingerlings. A total of 405 fish (6.88 ± 0.90 g) were exposed to varying concentrations of clove oil (25, 50, 75, 100 mg L⁻¹), propofol (10.0, 12.5, 25.0, 50 mg L⁻¹), or no anesthetic (control) in triplicate. Hematocrit and monocyte levels remained unchanged across treatments, but the highest doses of both anesthetics significantly reduced leukocyte percentages (p = 0.049 and p = 0.043, respectively). While serum enzymes were stable, cortisol levels increased significantly at the highest concentrations of both clove oil (38.92 ± 5.1 ng mL⁻¹; p = 0.001) and propofol (36.48 ± 3.4 ng mL⁻¹; p = 0.035), indicating secondary stress responses. Propofol at 25 mg L⁻¹ showed fast induction and recovery times and induced milder stress compared to clove oil. Overall, short-term anesthesia with both agents triggered physiological stress, but propofol at 25 mg L⁻¹ appeared more suitable for minimizing adverse effects in Persian sturgeon fingerlings.

1. Introduction

Sturgeons have lived almost unchanged for approximately 200 million years, despite major environmental changes over time [1]. However, in the 20th century, the wild sturgeon populations have critically declined and remain threatened for several reasons, including habitat destruction, migratory barriers, water pollution, exploitation, poaching, and illegal trade for their meat and caviar [2]. Persian sturgeon, Acipenser persicus (Borodin, 1897), is one of the six sturgeon species inhabiting the southern margin of the Caspian basin, and caviar is the main reason for its exploitation. A. persicus is an anadromous species, migrating from brackish water (Caspian Sea), where they spend most of their lives, to freshwater for spawning. This species, like other sturgeon and paddlefish species, is at risk of extinction and was listed in CITES Appendix II in 1998 as a highly endangered species. Therefore, aquaculture practices were developed to rescue wild stocks of A. persicus from extinction in the Caspian Sea by developing artificial propagation methods, restocking programs, and rearing systems [1,3].

The application of anesthesia is a commonly accepted procedure in aquaculture operations [4,5,6,7] for surgery, biopsy, blood sampling, sorting, handling, tagging, and reducing other stressors during aquaculture operations. Clove oil, extracted from the dried flower buds of Syzygium aromaticum, is widely used as a natural anesthetic in aquaculture due to its efficacy, availability, and low cost. Clove oil has been applied successfully in several sturgeon species, including Russian, Persian, and Siberian sturgeons, showing good anesthetic performance [8,9,10,11,12,13,14,15]. Its active compound, eugenol (4-allyl-2-methoxyphenol), possesses antibacterial, antifungal, and antioxidant properties [16,17,18,19,20,21,22]. However, concerns remain regarding its safety: eugenol can accumulate in fish tissues and may pose risks to farm personnel. Additionally, its use in food fish is restricted in some regions due to toxicological findings related to isoeugenol, a closely related compound. These limitations underscore the need to explore alternative anesthetic agents with better safety profiles and regulatory acceptance. Indeed, the US Food and Drug Agency has not approved a commercial anesthetic (Aqui-S, Scan Aqua, Norway) containing isoeugenol (a main bioactive component of clove oil) due to the evidence of cancer in the exposed male mice [23].

Propofol (2,6-diisopropylphenol) is used in both human and veterinary medicine for the induction and maintenance of sedation and general anesthesia. This can be accomplished in fish by intravenous injection or immersion procedures [24,25]. The usefulness of propofol for fish anesthesia has been studied in several fish species [9,26,27,28,29,30,31,32,33]. Adel et al. [4] reported the effect of four anesthetic agents (i.e., clove oil, propofol, phenoxyethanol, and ketamine) on induction and recovery times, and the mortality rate, in juvenile Persian sturgeon. Hematological and serum biochemical profiles are a vital tool in evaluating the general health status of Persian sturgeon, but the impact of different anesthetic agents on hematology and biochemistry is unknown.

Despite the availability of several anesthetics for aquaculture, there is a lack of comparative data on their physiological impacts on Persian sturgeon (A. persicus), an endangered species of high commercial value. In particular, while both clove oil and propofol are used in other fish species, their differential effects on stress-related hematological and biochemical parameters in A. persicus fingerlings remain unclear. Addressing this gap is crucial to optimizing anesthesia protocols that minimize physiological stress in aquaculture practices. Therefore, the aim of this research was to compare the effects of clove oil and propofol on physio-metabolic responses in Persian sturgeon fingerlings. It was hypothesized that propofol is ideal when short-term anesthesia is needed in Persian sturgeons, as it minimizes the possible side effects on health biomarkers. Additionally, it is important to consider that acquiring propofol can be significantly more challenging than obtaining eugenol, which may influence the choice of anesthetic in practical applications.

2. Materials and Methods

2.1. Fish Acclimation in Laboratory

A. persicus fingerlings (n = 405) were obtained from the Shahid Rajaie hatchery and culture center in Sari, Mazandaran (Iran), and transported in aerated tanks to the laboratory of the Research Center of Caspian Sea, Sari, Iran. At the start of the study, fish were examined for health. The fish were acclimated to the new environment in 200 L containers, with a flow rate of 5 L min⁻¹ for two weeks. They were fed a commercial sturgeon pellet three times a day, apparently satiated (Kimiyagaran-e Taghziyeh Company, Shahr-e Kord , Iran, including 40% crude protein, 12% crude lipid, 10% ash, and 8% moisture). The water parameters were monitored daily and maintained at 21.5 ± 1.3 °C, dissolved oxygen at 7.3 ± 0.5 mg L⁻¹, pH 7.8 ± 0.26, total hardness of 287.6 ± 4.3 mg L⁻¹, and salinity 9.4 ± 0.23‰ by daily exchanging the water. The natural photoperiod (11L:13D) was followed throughout the experiment. The fish were fasted for 24 h before the start of each test. This study was conducted according to the National/International principles of the Ethical Committee for the Use and Protection of Animals in scientific studies.

2.2. Anesthetic Agents

The essential oil of clove flower buds (Eugenia caryophyllus, syn. Syzygium aromaticum, Barijessence Pharmaceutical Co., Kāshān, Iran, 84% eugenol) was used. The stock solutions of clove oil were prepared a few minutes before use by dissolving the essential oil in 96% ethanol at 1:10 ratios. Propofol (Rapinovet, Fresenius Kabi, Austria) was directly added to the experimental tanks.

2.3. Experimental Design

A total of 405 A. persicus fingerlings were randomly divided into nine groups with 45 fish per group, and these groups were further divided into three replicates (15 fish in each tank). The mean initial fish weight (6.88 ± 0.90 g) and total length (110.6 ± 28.2) were not significantly different among the experimental groups. The fish in each subgroup were subjected to the corresponding anesthetic solutions to induce anesthesia. The concentrations of clove oil and propofol were 25, 50, 75, and 100 mg L⁻¹, and 10.0, 12.5, 25.0, and 50.0 mg L⁻¹, respectively. The control group was exposed to anesthetic-free water. The time taken to reach stage II of anesthesia and the recovery time were recorded using a digital stopwatch for each treatment [34]. The key features of stage II were loss of equilibrium and muscle tone, no reaction to touch stimuli, and decreased respiratory rate [35]. As soon as the fish reached stage II of anesthesia, they were removed from the anesthetic solution, rinsed quickly with water, and blood samples were collected. After the blood collection, the fish were transferred to the tanks containing freshwater without an anesthetic agent, equipped with gentle aeration (7.3 mg L⁻¹ dissolved oxygen) to be recovered. In the recovery tanks, the fish were individually placed in a 100 L tank and monitored to ensure the oxygenated water flow passed over the gills.

2.4. Blood Sampling and Hematological Assays

All the fish (n = 15) in each tank were treated with the specific anesthetic agent at certain concentrations, and then, nine fish were randomly caught with a hand net as soon as they reached stage II of anesthesia. Blood was immediately withdrawn from the caudal vein with a sterile syringe (nine fish from each anesthesia-treated group). The blood samples (n = 5) from each tank (replicate) were pooled and immediately divided into two equal volumes. One sample was transferred to a tube coated with heparin (lithium heparin; Pars Peyvand Co., Tehran, Iran) for hematological analyses, while the other sample was transferred to nonheparinized tubes for serum biochemical studies. Serum samples were obtained by the centrifugation method (3000× g for 15 min) and stored at −80 °C until analysis. The total number of red blood cells (RBC) and white blood cells (WBC) was counted in a Neubauer hemocytometer using Hayem and Türck diluting fluids [36]. Hematocrit (Ht) was determined by the standard microhematocrit method. Hemoglobin (Hb) concentration was determined according to the cyanomethemoglobin procedure. Differential leukocyte count was determined from Giemsa-stained smears under light microscopy (Nikon Eclipse 80i Nikon, Melville, NY, USA).

2.5. Serum Biochemical Analysis

Glucose, triglycerides, total protein (TP), albumin, globulin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) levels were estimated from serum samples using standard curves from the relevant commercial kits (Pars Azmoon Company, Tehran, Iran) and a biochemical auto analyzer (Eurolyser, Salzburg, Austria). Both methods have previously been applied to samples obtained from fish [37]. The cortisol (ng mL⁻¹) concentration was determined with a commercial ELISA kit (DRG Diagnostics, Mountainside, NJ, USA) based on the assay described by Shaluei et al. [38].

2.6. Statistical Analysis

Data normality (Kolmogorov–Smirnov test) and homogeneity of variances (Levene’s test) were assessed. Data were normally distributed and reported as mean ± standard deviation (SD). Data were analyzed with ANOVA. To detect significant differences among hematological and serum biochemical parameters in different groups, the means were subjected to Duncan’s multiple-range test. In all statistical analyses, values of p < 0.05 were considered significant. A power regression model was used against propofol and clove oil at different concentrations as fixed factors. Statistical analyses were conducted using commercial software (SPSS software version 22, IBM Corp., Chicago, IL, USA).

3. Results

3.1. Induction and Anaesthetic Recovery

The time to achieve stage II anesthesia was dose-dependent (Figure 1). Propofol induced anesthesia in a significantly shorter time than clove oil at 25 and 50 mg L⁻¹. However, the recovery time was prolonged (>10 min) in fish treated with clove oil at ≥50 mg/L (Figure 1 and

Table 1. Mean induction and recovery times (in minutes, ±SD) of Persian sturgeon (A. persicus) fingerlings exposed to increasing concentrations of clove oil and propofol.

Concentration (mg/L)	Induction Time (min)		Recovery Time (min)
	Clove Oil	Propofol	Clove Oil	Propofol
10	—	3.56 ± 0.21	—	6.23 ± 0.30
12.5	—	3.39 ± 0.18	—	7.45 ± 0.42
25	7.08 ± 0.44	2.38 ± 0.16	5.75 ± 0.27	8.10 ± 0.38
50	2.15 ± 0.15	1.71 ± 0.13	10.74 ± 0.40	9.37 ± 0.33
75	1.61 ± 0.12	—	11.38 ± 0.48	—
100	1.11 ± 0.09	—	11.41 ± 0.52	—

Data represent the time required to reach stage II anesthesia (loss of equilibrium and no response to external stimuli) and the subsequent recovery period, recorded immediately after anesthetic exposure. Each value is based on triplicate trials (n = 3) per treatment group.

).

3.2. Hematological Indices

Clove oil anesthesia did not significantly affect the RBC count (F = 0.166, p = 0.951) and HCT concentration (F = 3.182, p = 0.063; Table 1). However, fish exposed to the highest dose of clove oil showed a significant decrease in the Hb concentration (F = 6.020, p = 0.010; Table 1). Moreover, WBC decreased significantly in fish exposed to clove oil at 50, 75, and 100 mg L⁻¹ compared to the control and 25 mg L⁻¹ clove oil groups. The lymphocyte percentage followed a decreasing trend as clove oil concentration increased, and the lowest value was obtained in the 75 and 100 mg L⁻¹ clove oil groups. However, the neutrophil percentage was inversely associated with clove oil concentration, and the highest level was recorded in the groups exposed to clove oil at 50–100 mg L⁻¹ compared to the control fish (F = 5.472, p = 0.013;

Table 2. Effects of clove oil at various concentrations on hematological indices in Persian sturgeon (A. persicus) fingerlings.

Indicator	Clove Oil Concentration (mg L−1)
	0 (Control)	25	50	75	100
RBC (×106 mL−1)	0.86 ± 0.08 a	0.87 ± 0.09 a	0.84 ± 0.06 a	0.85 ± 0.07 a	0.83 ± 0.10 a
Hb (g L−1)	6.72 ± 0.44 a	6.60 ± 0.32 a	6.48 ± 0.40 a	6.25 ± 0.38 ab	5.94 ± 0.25 b
HCT (%)	21.2 ± 0.7 a	21.1 ± 0.4 a	21.4 ± 0.5 a	20.3 ± 0.4 a	20.5 ± 0.8 a
WBC (×103 mL−1)	23.16 ± 0.26 a	22.84 ± 0.22 a	22.26 ± 0.18 b	22.34 ± 0.15 b	22.20 ± 0.16 b
Lymphocytes (%)	83.8 ± 1.1 a	83.4 ± 1.0 ab	82.9 ± 0.8 ab	81.5 ± 1.2 b	81.8 ± 0.9 b
Neutrophils (%)	9.1 ± 0.6 b	9.5 ± 0.7 ab	11.0 ± 0.9 a	11.6 ± 0.9 a	11.2 ± 0.9 a
Monocytes (%)	4.2 ± 0.5 a	4.0 ± 0.6 a	4.3 ± 0.4 a	4.1 ± 0.5 a	3.9 ± 0.6 a
Eosinophil (%)	1.9 ± 0.2 a	1.7 ± 0.3 a	1.8 ± 0.1 a	2.0 ± 0.4 a	2.1 ± 0.3 a

Data are presented as mean ± SD. Values within a row with different superscript letters are significantly different (p < 0.05). RBC, red blood cells; Hb, hemoglobin concentration; HCT, hematocrit; WBC, white blood cells.

).

An increasing trend was observed in RBC, Hb, and HCT values by increasing the propofol concentrations up to 12.5 mg L⁻¹ and then decreasing, while the differences among the groups were not significant (F > 0.639, p > 0.130; Table 2). Exposure to 50 mg L⁻¹ propofol significantly decreased the WBC count among the experimental treatments (F = 6.569, p = 0.007; Table 2), which was followed by a significant reduction in the leucocyte count (from 83.5% in the control group to 80.6% in the propofol anesthetized group; F = 3.675, p = 0.043). The apparent increase in the neutrophil percentage was found in the anesthetized fish with the 50 mg L⁻¹ propofol group (F = 4.57, p = 0.023;

Table 3. Effects of propofol various concentrations on hematological indices in Persian sturgeon (A. persicus) fingerlings.

Indicator	Propofol Concentration (mg L−1)
	Control	10.0	12.5	25.0	50.0
RBC (×106 mL−1)	0.85 ± 0.07 a	0.87 ± 0.07 a	0.90 ± 0.09 a	0.86 ± 0.06 a	0.80 ± 0.08 a
Hb (g L−1)	6.70 ± 0.45 a	6.87 ± 0.62 a	7.23 ± 0.40 a	7.02 ± 0.48 a	6.64 ± 0.65 a
HCT (%)	21.2 ± 0.2 a	21.3 ± 0.3 a	22.0 ± 0.6 a	20.7 ± 0.8 a	20.9 ± 0.7 a
WBC (×103 mL−1)	23.2 ± 0.3 a	23.3 ± 0.4 a	23.1 ± 0.2 a	22.9 ± 0.3 a	22.0 ± 0.2 b
Lymphocytes (%)	83.5 ± 1.1 a	83.2 ± 1.0 a	82.8 ± 1.2 a	82.6 ± 0.9 a	80.6 ± 0.8 b
Neutrophils (%)	9.4 ± 0.7 b	9.7 ± 0.6 b	10.2 ± 0.8 b	10.0 ± 0.7 b	11.5 ± 0.9 a
Monocytes (%)	3.9 ± 0.4 a	3.8 ± 0.5 a	4.0 ± 0.7 a	4.2 ± 0.6 a	4.0 ± 0.3 a
Eosinophil (%)	2.0 ± 0.3 a	2.1 ± 0.4 a	2.2 ± 0.5 a	1.9 ± 0.3 a	2.1 ± 0.6 a

Data are presented as mean ± SD. Values in the raw with different letters are significantly different (p < 0.05; n = 3). RBC, red blood cells; Hb, hemoglobin concentration; HCT, hematocrit; WBC, white blood cells.

).

3.3. Serum Biochemical Parameters

A significant increase in cortisol occurred during exposure to clove oil, and the highest levels were obtained at the highest doses (p = 0.001 and p = 0.035, respectively;

Table 4. Effects of clove oil various concentrations on biochemical parameters in Persian sturgeon (A. persicus) fingerlings.

Indicator	Clove Oil Concentration (mg L−1)
	Control	25	50	75	100
Glucose (mmol L−1)	2.49 ± 0.15 b	2.79 ± 0.36 b	3.58 ± 0.25 a	3.31 ± 0.22 a	3.45 ± 0.23 a
TP (g L−1)	25.8 ± 2.4 a	25.2 ± 1.9 a	23.4 ± 1.8 a	23.8 ± 2.0 a	20.4 ± 1.3 b
TGs (mmol L−1)	66.0 ± 9.8 a	69.2 ± 10.6 a	65.4 ± 9.1 a	70.1 ± 10.4 a	68.3 ± 10.6 a
ALT (U L−1)	11.58 ± 1.2 a	11.74 ± 1.0 a	11.62 ± 1.2 a	11.43 ± 1.5 a	11.87 ± 0.9 a
AST (U L−1)	218.3 ± 26.2 b	274.5 ± 30.6 b	320.6 ± 34.9 a	282.1 ± 32.3 ab	308.4 ± 35.0 a
LDH (U L−1)	1342 ± 119 a	1376 ± 125 a	1369 ± 120 a	1394 ± 132 a	1329 ± 111 a
Cortisol (ng mL−1)	18.32 ± 1.8 c	27.42 ± 2.9 b	25.82 ± 2.3 b	29.87 ± 4.7 b	38.92 ± 5.1 a

Data are presented as mean ± SD. Values in the raw with different letters are significantly different (p < 0.05; n = 3). TP, total protein; TGs, triglycerides; ALT, alanine aminotransferase; AST, aspartate aminotransferase; LDH, lactate dehydrogenase.

).

Glucose levels were significantly increased with exposure to clove oil at 50–100 mg L⁻¹ (F = 9.188, p = 0.002) and propofol at 25 mg L⁻¹ compared to the control group (F = 4.026, p = 0.034; Table 3 and Table 4). Exposure to 100 mg L⁻¹ clove oil led to a significant decrease in the total protein concentration compared to other concentrations (F = 3.632, p = 0.045; Table 3). No significant difference was observed in total protein concentration in the propofol treatment groups (F = 0.343, p = 0.843; Table 3). Exposure to clove oil at 50 and 100 mg L⁻¹ caused a significant increase in the serum AST level (F = 4.560, p = 0.024; Table 3). No significant changes in the serum enzymes were found in the propofol treatment groups (F > 0.167, p > 0.814;

Table 5. Effects of propofol various concentrations on biochemical parameters in Persian sturgeon (A. persicus) fingerlings.

Indicator	Propofol Concentration (mg L−1)
	Control	10.0	12.5	25.0	50.0
Glucose (mmol L−1)	2.49 ± 0.19 b	2.67 ± 0.25 ab	2.79 ± 0.21 ab	3.40 ± 0.44 a	2.92 ± 0.33 ab
TP (g L−1)	26.0 ± 1.4 a	26.4 ± 1.6 a	25.7 ± 1.3 a	26.8 ± 1.9 a	25.6 ± 1.2 a
TGs (mmol L−1)	66.0 ± 10.4 a	67.4 ± 12.3 a	70.9 ± 13.6 a	69.2 ± 13.4 a	70.3 ± 14.5 a
ALT (U L−1)	11.72 ± 1.0 a	11.86 ± 1.4 a	11.52 ± 1.7 a	11.64 ± 1.5 a	12.04 ± 1.6 a
AST (U L−1)	219.7 ± 20.8 a	228.5 ± 24.0 a	235.1 ± 30.2 a	242.3 ± 27.6 a	240.2 ± 28.5 a
LDH (U L−1)	1376 ± 128.2 a	1392 ± 135.4 a	1435 ± 140.0 a	1412 ± 135.8 a	1490 ± 156.6 a
Cortisol (ng mL−1)	17.92 ± 1.6 c	22.18 ± 2.3 c	20.76 ± 2.7 c	28.14 ± 3.9 b	36.48 ± 3.4 a

Data are presented as mean ± SD. Values in the raw with different letters are significantly different (p < 0.05; n = 3). TP, total protein; TGs, triglycerides; ALT, alanine aminotransferase; AST, aspartate aminotransferase; LDH, lactate dehydrogenase.

).

4. Discussion

To our knowledge, this is the first study to provide a side-by-side evaluation of clove oil and propofol effects on hematological and biochemical stress markers in Persian sturgeon fingerlings. This offers practical guidance for selecting the most appropriate anesthetic in aquaculture settings for this endangered species.

In 1985, following 50 years of experience from different researchers, Marking and Meyer developed criteria to reach stage II of anesthesia in fish using anesthetics, which includes an induction time of less than 3 min, a recovery time shorter than 10 min, and no mortality within 48 h after the exposure [39]. According to Bell [40], if the induction time of anesthesia is shorter than 2 min, the anesthesia should be immediately stopped because the concentration can be dangerous for fish health. The present findings, according to the study of Adel et al. [9], refine existing knowledge on anesthesia protocols in A. persicus, building on previous studies by providing a direct comparison of clove oil and propofol in terms of both anesthetic efficiency and physiological impact. It can be estimated that the optimum anesthetic concentrations for Persian sturgeon fingerlings ranged from 45 to 60 mg L⁻¹ and from 15 to 36 mg L⁻¹ for clove oil and propofol, respectively. In contrast to propofol, the recovery time of clove oil at the optimum induction concentrations exceeded 10 min in Persian sturgeon [9]. Arani et al. [11] also found that both induction time and recovery time became shorter by increasing Persian sturgeon weight (from 1.89 g to 4.25 g) when exposed to clove oil anesthesia. The prolonged recovery time can be due to the longest anesthesia durations in the gills, as the main path of the anesthetic excretion, based on the physicochemical properties of the anesthetic [41,42]. On the other hand, a greater concentration of anesthetics leads to a higher rate of anesthetic absorption in fish [43]. Our data suggest that propofol, due to its rapid induction and limited impact on serum biomarkers at moderate doses, may represent a more suitable anesthetic for short procedures in juvenile A. persicus.

Hematological indices are commonly used as promising biomarkers in fish to elucidate psychological changes in response to internal and external stressors [44,45,46,47,48,49,50]. Clove essential oil and propofol are lipophilic, and they rapidly enter through the gill epithelium and cross the blood–brain barrier, resulting in a rapid onset of reactions [51,52]. Therefore, evaluating the blood physio-metabolic profiles regarding the anesthetics in Persian sturgeon can be useful even in short-term exposure to anesthetics. Based on the findings from the present study, although some hematological values (RBC and Hb) differed significantly in anesthetized fish, it can be concluded that these changes in clove oil and propofol anesthesia did not indicate any specific disease (e.g., anemia) and compromise health status in Persian sturgeon fingerlings. In sturgeons, RBC count is typically in the lower range of the reference intervals for fish (0.4 to 5.2 × 10⁶/mm³) [53] due to their lower mobility as a result of lower oxygen demand. Gomulka et al. [13] found a significant drop in RBC count in Siberian sturgeon, Acipenser baerii (Brandt, 1869) (94.9 g mean body weight), exposed to 79.5 mg L⁻¹ of eugenol for 10 min. Moreover, Russian sturgeon, Acipenser gueldenstaedtii (Brandt & Ratzeburg, 1833) (116.3 g mean weight), was exposed to propofol (10 mg L⁻¹) and eugenol (42.4 mg L⁻¹) for 10 min and revealed only a slight increase in HCT and Hb values. The changes in RBC count are probably the consequence of regulatory mechanisms, which maintain RBC internal pH [54] during respiratory acidosis following prolonged anesthesia [26].

Cortisol initiates lymphocyte apoptosis [55]. The decreased lymphocyte count accompanied by an increase in the neutrophil percentage usually follows acute stress in fish [56]. Leukogram in fish, like other vertebrates, includes WBC and differential leukocyte count, which is related to the immune system, and the alterations can profoundly reflect the health status in response to various stress factors [57]. In this study, the exposure of Persian sturgeon to the highest dose of propofol (50 mg L⁻¹) resulted in a significant increase in the serum cortisol level, while the lowest lymphocyte percentage was obtained in this dose. In the case of clove oil-exposed fish, the notably increased cortisol level was recorded in 100 mg L⁻¹ clove oil, while the lymphocyte percentage was significantly decreased in 75 and 100 mg L⁻¹ clove oil treatments compared to the control fish. No effect of clove oil on lymphocyte count was found immediately after anesthesia in Russian sturgeon (A. gueldenstaedtii), Siberian sturgeon, rainbow trout (Oncorhynchus mykiss), and common carp (Cyprinus carpio) [58,59], while the lymphocyte percentage was markedly decreased 24 h after the exposure of Persian sturgeon to propofol at 50 mg L⁻¹ and clove oil at 75 and 100 mg L⁻¹.

It has been proved that the serum glucose level is a predictable marker of stress handling in fish. According to Barton [60], a rapid release of catecholamines is the main cause of increased blood glucose and usually occurs in fish under acute stress. Prolonged stress can lead to an induction of gluconeogenesis and depletion of total protein levels in fish blood. In general, hyperlipidemia can be induced by releasing adrenaline from chromaffin tissue [9], which is an alternative to the corticoid stress response pathway and allows for the mobilization of energy stores in fish. Both clove oil and propofol did not markedly affect the triglyceride levels in the exposed Persian sturgeon, probably due to the relatively short exposure time. However, the glucose level was significantly increased in the fish exposed to 50, 75, and 100 mg L⁻¹ of clove oil and 25 mg L⁻¹ of propofol. An increase in the serum cortisol raises blood glucose levels (secondary stress response) by glycogenolysis to compensate for the amount of energy needed to overcome stress [61,62]. Previous studies have declared that anesthetics can inhibit the activation of the hypothalamus–pituitary–interrenal (HPI) axis in fish and ultimately prevent the secretion of cortisol [63]. Therefore, propofol is probably more potent in reducing the HPI axis activation in Persian sturgeon compared to clove oil. There are three cascades of reaction in fish to react against stressors, including primary, secondary, and tertiary responses [60]. Based on the obtained physio-metabolic changes, although the primary stress response (high cortisol level) of Persian sturgeon fingerlings exposed to clove oil and propofol was similar, the secondary (hyperglycemia) and tertiary (lymphopenia; suppression of the immune system) stress responses were more moderate in the case of propofol-exposed fish. We cannot assign whether the stress was induced due to the chemical compounds of the anesthetics or behaviorally mediated. Since the chemical structure of clove oil and propofol are different, but both of them led to a significant cortisol release in the highest doses, the latter explanation seems more likely. A similar interpretation was given for Zebrafish when they were exposed to different anesthetics [64]. However, further studies are needed to address pharmacokinetics and pharmacodynamics models in fish in response to different anesthetics.

ALT and AST are intracellular enzymes, and their increased levels in fish serum usually suggest hepatic tissue damage [65]. The propofol exposure did not cause any changes in the serum ALT and AST levels, but the fish exposed to clove oil (50 and 100 mg L⁻¹) had a more than 20% increase in the serum AST compared to the propofol-anesthetized fish. There is a strong relationship between liver health status and the synthesis of proteins (chiefly albumin and globulins) into the bloodstream [66]. An increase in the serum AST in clove oil treatments (50 and 100 mg L⁻¹) compared to the control fish may indicate liver damage in Persian sturgeon that can justify the notable reduction in serum TP in the fish exposed to 100 mg L⁻¹ clove oil. While both anesthetics induced moderate secondary stress responses, propofol demonstrated a more favorable profile overall—particularly at 25 mg L⁻¹—making it a promising candidate for routine use in hatcheries and restocking programs involving Persian sturgeon. The probability of tertiary stress responses and liver damage is lower in the case of propofol anesthesia when compared to clove oil.

These physiological responses should be interpreted in the context of fish welfare and the operational demands of aquaculture, where rapid induction, recovery, and minimal disruption to homeostasis are crucial. However, further investigations are needed to elucidate the effects of propofol and clove oil on liver histology and the antioxidant defense system in fish under short and long-term anesthesia exposure due to the limited financial resources, time, and samples in this study.

5. Conclusions

We can conclude that both clove oil and propofol short-term anesthesia caused similar moderate secondary stress responses in Persian sturgeon fingerlings. The probability of a tertiary stress response is low in the group treated with propofol anesthesia when compared to clove oil. However, there is a need for a survey on the toxicity and histopathological changes in clove oil and propofol in Persian sturgeon during short and prolonged exposure. In future studies, it is also highly recommended that the physio-metabolic changes be determined after a longer time from the recovery stage to elucidate the long-term effects of the anesthetics in Persian sturgeon.