Five experimental diets were created to meet the specific dietary needs of fingerlings of Nile tilapia, all of which were isonitrogenous and had the same amount of calories. Five groups were assigned different diets: Group 1 (G1, HEOs 0 mL/kg) received no chemicals, and Groups 2 (G2, HEOs 30 mL/kg) and 3 (G3, HEOs 60 mL/kg) received 30 and 60 mL of herbal essential oils (HEOs) per kilogram of feed, respectively. Supplementation levels were increased for Groups 4 (G4, HEOs 120 mL/kg) and 5 (G5, HEOs 240 mL/kg) to 120- and 240-ml HEOs/kg of diet, respectively—Supplementary Table S1 - GC–MS Composition of HEO Blend.

The experimental diets were formulated to be isonitrogenous and isolipidic, with a target of 32% crude protein and 7% lipid. All ingredients were dried to constant weight and mixed on a dry matter (DM) basis. Proximate composition was determined according to AoOA Chemists and AoOA Chemists (19) methods: crude protein by the Kjeldahl method (Method 2001.11), crude lipid by ether extraction (Method 920.39), ash by incineration at 550°C (Method 942.05), and moisture by oven drying at 105°C (Method 930.15).

The basic elements of the diet include fish meal (the main source of animal protein), wheat bran, rice bran, wheat flour, soybean meal, corn gluten, yellow maize, di-calcium phosphate, fish oil, limestone, and a premixed vitamin and mineral blend. Five equal quantities of the materials were measured for each treatment after thorough mixing Table 1.

The liquid herbal essential oils (TRI VIR, Tri Pharma Company, Egypt) were added to each treatment diet in a specific dosage. These oils included carvacrol (45 g/L), oregano oil (45 g/L), 1,8-cineole (16 g/L), thymol (39.2 g/L), α-pinene (4.6 g/L), β-pinene (2.6 g/L), limonene (3 g/L), and propylene glycol (150 g/L). Each batch was mixed with fish oil after the HEOs were added, and then 400 cc of water per kilogram of feed was added to make a homogenous dough. To make pellets that are just right for fingerlings, this dough was run through laboratory pelleting equipment fitted with a 2 mm die. The pellets were left to dry at room temperature for a full day before being sealed in plastic bags and kept at 4°C until needed. We used industry-standard methods to examine the diets’ chemical makeup (20).

Nile tilapia were collected from a freshwater farm with no prior health issues or mortality. Fish were sedated onsite using MS-222 (40 mg/L, Syndel, Canada), then transported in labeled plastic bags containing one-third clean water and two-thirds pure oxygen. Upon arrival at the Animal Health Research Institute, Agriculture Research Center, Kafrelsheikh, Egypt, fish underwent a 10-min iodine bath (20 ppm Betadine®, 5% povidone-iodine, Nile Company for Pharmaceuticals) (21).

At first, the Nile tilapia fingerlings were kept in a 1,000-liter fiberglass tank at the Sakha Aquaculture Research unit after being acquired from a private fish farm in Kafrelsheikh, Egypt. Fifteen days before the trial’s start, the fish were given a commercial meal with 30 % crude protein to help them adjust to the lab environment. After acclimatization, 300 fingerlings (27.20 ± 0.06 g) were accidentally allocated to 30 glass aquaria (each with a capacity of 60 liters), with 10 fish stocked per tank. Three aquaria were designated for each dietary treatment, and all tanks were continuously aerated using electric air pumps. The photoperiod was maintained at 12 h light and 12 h dark using programmable LED lighting. Initial biomass density was approximately 4.5 kg/m³ (10 fish averaging 27.2 g per 60-L tank). Dissolved oxygen was measured daily at 9:00 a.m. and 5:00 p.m. using a DO meter (YSI Professional Plus), and ranged from 6.5 to 7.0 mg/L across all tanks. Tank positions were randomized at the beginning of the experiment using a computer-generated random number sequence and re-randomized weekly to eliminate potential positional or environmental bias (light, airflow). Dechlorinated water was used throughout the study. Waste was removed daily by siphoning, with 50% of the water in each tank replaced daily, and a full water change was carried out once weekly following cleaning. Fish were fed the experimental diets twice daily—at 8:00 a.m. and 2:00 p.m.—for 72 days. Feeding was based on 3% of the fish’s body weight and was adjusted biweekly according to growth. Water quality parameters were regularly monitored, with the following average values recorded: temperature at 28.53 ± 0.01°C, dissolved oxygen at 6.73 ± 0.14 mg/L, pH at 7.62 ± 0.1, and total ammonia nitrogen at 0.38 ± 0.02 mg/L.

The duration of the feeding trial was 72 days. Each fish in each tank was weighed before and after the experiment to see how much they had grown. The fish were fasted for 24 h before the final sampling to reduce stress. The following equations were used to assess the efficiency of feed use and growth performance:

Prior to sampling, fish were fasted for 24 h to eliminate postprandial effects on hematological and biochemical parameters. Blood was collected from the caudal vein using 1 mL heparinized syringes under anesthetic conditions.

Fish blood was drawn from the caudal vertebral vein at the end of the experiment using sterilized hypodermic syringes and an anesthetic. Blood samples were separated into two tubes. Hematological tests were performed in the first tube using EDTA as an anticoagulant. The second tube without an anticoagulant was left at room temperature for 3 h to allow clot formation. After centrifuging the clotted blood at 3000 RPM for 15 min, serum was extracted. The serum was stored at −20°C until biochemical analysis.

The Quantitative gene expression analysis was done using real-time reverse transcription PCR (RT-PCR). The genes EF1-α and β-actin served as internal controls for data normalization (primer sequences are provided in Table 2). Total RNA was isolated from tissue samples using Trizol reagent (iNtRON Biotechnology), following the manufacturer-supplied protocol. The integrity of RNA was confirmed through electrophoresis on a 2% agarose gel, and concentrations were determined using a Nanodrop spectrophotometer (Quawell, United States).

To synthesize cDNA, 2 μg of high-quality total RNA from each sample was reverse transcribed using a Bioline cDNA synthesis kit (United Kingdom), adhering to the manufacturer’s recommendations. PCR amplification was conducted using the SensiFast SYBR Lo-Rox kit (Bioline) in a 20 μL total volume. Each reaction included 2 μL of cDNA template, 10 μL of SYBR Green master mix, and 0.5 μM of each primer.

The thermal cycling program included an initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 s and annealing/extension at 60°C for 1 min. Amplification data and Ct values were collected automatically using the Rotor-Gene Q system (Qiagen, Valencia, CA, United States). The relative quantification of target gene expression was calculated using the 2^−ΔΔCt method (31). To validate the stability of the two reference genes (EF1-α and β-actin), expression data were analyzed using **geNorm** and **NormFinder** software tools. The geNorm analysis revealed M-values of 0.41 (EF1-α) and 0.44 (β-actin), both below the accepted threshold of 1.5, indicating stable expression under our experimental conditions. NormFinder also ranked these genes as the two most stable among the four candidates tested (including GAPDH and 18S rRNA; data not shown), with a combined stability value = 0.28. Primer efficiency was calculated from standard curves generated by serial 10-fold dilutions of pooled cDNA. All primer pairs exhibited efficiencies between **95 and 104%** and R² values > 0.99.

Five randomly selected fish from each treatment (HEOs 0, 30, 60, 120, and 240 mL/kg) were histopathologically examined following deep anesthesia with 40% ethyl alcohol. Tissue samples were taken from the front, middle, and posterior intestines. The materials were dehydrated in 70–100% ethyl alcohol after a one- to two-day fixation in 10% formaldehyde. After dehydration, samples were paraffin waxed and xylene washed. According to histopathological and morphometric analysis techniques, Hematoxylin and eosin staining were performed on 4–5 μm sections (32, 33). Image analysis software (NIH, Bethesda, MD) evaluated intestinal villi length, breadth, and crypt depth. Each set of five intestinal cross-sections had 10 randomly selected villi and crypts, which were examined using one-way ANOVA (SPSS version 22, SPSS Inc., IL, United States). A p-value under 0.05 was significant. Although five fish per treatment were histologically examined, we confirm that three non-overlapping intestinal sections per fish were evaluated to increase statistical robustness. This results in 15 observations per treatment group for morphometric analysis.

All statistical analyses were conducted using SPSS version 22 (IBM, United States). The experimental unit for all analyses was the tank (n = 3 per treatment), and tank-level means were used to avoid pseudoreplication.

Data were first checked for normality using the Shapiro–Wilk test and for homogeneity of variances using Levene’s test. When assumptions were met, one-way analysis of variance (ANOVA) was used to detect differences among treatments, followed by means were compared using the Tukey–Kramer post hoc test to account for unequal sample sizes. The level of significance was set at α = 0.05.

In addition, a linear mixed model (LMM) was fitted using the `PROC MIXED` procedure to account for random tank effects. The model equation was:

Where Y_ij is the observed response in tank j of treatment i, μ is the overall mean, T_i is the fixed effect of treatment, and e_ij is the residual error (random). A variance components (VC) covariance structure was used. Model fit was assessed using Akaike Information Criterion (AIC) and residual plots. Residuals were confirmed to be normally distributed with homoscedasticity. For key response variables (e.g., SGR, FCR, GHr, IGF-I), both one-way ANOVA and LMM approaches yielded consistent results.

A total of 15 tanks (3 per treatment group) were used in the study. Post hoc power analysis was performed using G*Power version 3.1.9.7 to assess whether this design had sufficient sensitivity to detect significant treatment effects. Using a one-way ANOVA model with five groups, α = 0.05, and n = 3 replicates per group, the analysis showed that effect sizes of f = 0.40–0.50 (based on observed SGR and gene expression differences) yielded power levels >0.80. This indicates the sample size was sufficient for detecting large and biologically relevant treatment effects.

At the end of the 72-day feeding experiment, significant improvements in growth performance metrics were noted in Nile tilapia (p ≤ 0.05), as outlined in Table 3. The initial body weight (IBW) of fish across all treatment groups showed no statistical differences (p =0.654), indicating that all groups began the trial under equivalent conditions.

Tilapia-fed diets enriched with 30 or 60 mL/kg of herbal essential oils (HEOs) achieved the best growth responses, recording significantly higher values for final body weight (FBW), weight gain percentage (WG%), daily weight gain (DWG), feed intake (FI), feed efficiency (FE), and specific growth rate (SGR) compared to the other groups (p =0.0241).

On the other hand, the poorest growth outcomes were observed in the control group, and fish fed higher HEO inclusion levels (120 and 240 mL/kg). Feed conversion ratio (FCR) was significantly improved (i.e., reduced) in the 30 and 60 mL/kg HEO groups, whereas the control and high-inclusion groups did not show significant changes in FCR. Furthermore, both protein efficiency ratio (PER) and protein productive value (PPV) were substantially higher in the 30 and 60 mL/kg treatment groups than in the control or higher-dose groups (p =0.0145). Survival rates were also significantly better in the control group and those fed 30 and 60 mL/kg of HEOs, showing a noticeable decline in the groups receiving 120 and 240 mL/kg (p =0.0214).

Introducing HEOs substantially impacted blood hematological and biochemical parameters (p =0.0324). Table 4 shows that, associated with the control group, the Nile tilapia fish given a diet with 30, 60, 120, and 240 mL HEOs/kg−1 diet had increased levels of hemoglobin, platelet-count value (p =0.014), (RBCs), and (WBCs). Nevertheless, the current study did not find any significant impacts of utilizing a combination of HEOs on the blood biochemical features of Nile tilapia (p =0.0214; Table 5). Treatment groups also had reduced levels of triglycerides and cholesterol. As a result, they consumed less HEO in their diet compared to the control group and other groups, using a combination of 30 and 60 mL/kg−1 HEO (Table 5).

There were notable group variations in the fish immune response markers; those groups that consumed higher concentrations of HEO had an improved immune response (Table 6). In fingerlings, lysozyme and phagocytic activity showed a greater value. Unlike the other groups, Nile tilapia O. niloticus fed a diet containing 30 and 60 mL HEOs/kg−1 and fared relatively well. Several experimental meals had similar phagocytic index values (p =0.756). In the fish-fed diet with HEOs, IgM levels were substantially higher than in the control and fish-fed 60 mL HEOs/kg−1 diet (p < 0.05; Table 6).

Feeding Nile tilapia fingerlings with herbal essential oils (HEOs) significantly affected antioxidant enzyme activity (Table 6). Both (SOD) and (CAT) activities were substantially elevated (p ≤ 0.05) in fish-fed diets containing HEOs associated with the control group. The MDA levels showed no significant differences across all treatment groups (p =0.6547).

Regarding gene expression, the relative mRNA levels of HSP70 were substantially reduced in fish fed 30 and 60 mL/kg of HEOs (p =0.0145). In contrast, fish receiving 120 and 240 mL/kg diets exhibited a significant upregulation of HSP70 expression (Figure 1).

Fish fed 30 and 60 mL/kg HEOs showed lower IL-1β and TNF-α levels than other dietary groups (p =0.014; Figures 2A,B), indicating reduced inflammatory activity.

The expression of (GHr) in liver tissue was significantly upregulated in fish supplemented with 30 and 60 mL/kg of HEOs (Figure 3A), while ghrelin—a hormone associated with metabolism and appetite regulation—was also significantly more expressed in the stomachs of fish from these groups (p =0.017; Figure 3B).

Additionally, IGF-I gene expression in the liver was significantly higher in fish-fed diets containing 30 and 60 mL/kg of HEOs than in the other groups (p =0.025; Figure 4), suggesting enhanced growth signaling.

SOD and CAT were also significantly increased in the liver tissues of fish fed 30 and 60 mL/kg of HEOs (p ≤ 0.05; Figures 5A,B), consistent with the enzyme activity results. Zonula occludin-1 (ZO-1) and occludin showed significant increases in the groups receiving 30 and 60 mL/kg of HEOs, with the greatest expression detected in the 60 mL/kg group (p =0.036; Figures 6A,B), indicating improved gut barrier integrity.

Many intestinal villi are bordered by simple columnar epithelium containing goblet cells, and the intestinal wall seems intact in all three types. Their lamina muscularis and tunica serosa are external coverings for a core of loose connective tissue. Compared to other groups, the intestinal villi in the 30 mL/kg and 60 mL/kg HEOs groups exhibit high branching. Villi length, surface area, and villi length/crypt depth were all significantly increased in the 30 mL/kg and 60 mL/kg HEO groups compared to the 0 mL/kg, 120 mL/kg, and 240 mL/kg groups in the morphometric analysis of data acquired from the measurement of intestinal villi. In Table 7, you can see a summary of the results of the morphological analysis in Figure 7 and Supplementary Table S2.

To determine the optimal inclusion level of HEOs, broken-line and quadratic regression analyses were performed for specific growth rate (SGR), feed conversion ratio (FCR), and expression of GHr and IGF-I. The broken-line model estimated the optimal HEO inclusion level at 52.6 mL/kg diet for SGR and 55.4 mL/kg for FCR. Beyond this threshold, no further performance improvements were observed, and higher levels were associated with reduced gains.

These results are illustrated in Figure 8 and support our conclusion that the optimal HEO dose lies between 50 and 60 mL/kg.