Open Veterinary Journal, (2025), Vol. 15(3): 1424-1439

Short Communication

10.5455/OVJ.2025.v15.i3.33

The beneficial impact of oregano essential oil supplementation on growth, physiological function, antioxidant activity, and immune response in Nile tilapia (Oreochromis niloticus) fingerlings under Aeromonas hydrophila stress

Mohamed A. Khallaf¹, Wesam H. Marzouk², Hanan A. Ghetas¹, Haguer M. Salah El Din³, Mona Assas⁴, Azza Hafez⁵, Alaa Elgaabari⁶ and Eman M. Moustafa^3,*

¹Department of Aquatic Animals Medicine and Management, Faculty of Veterinary Medicine, University of Sadat City, Sadat City, Egypt

²Veterinarian at the General Organization for Veterinary Services in Cairo, Cairo, Egypt

³Fish Diseases and Management Department, Faculty of Veterinary Medicine, Kafrelsheikh University, Kafrelsheikh, Egypt

⁴Fish processing and Biotechnology Department, Faculty of Aquatic and Fisheries Sciences, Kafrelsheikh University, Kafrelsheikh, Egypt

⁵Nutrition and Clinical Nutrition Department, Faculty of Veterinary Medicine, Kafrelsheikh University, Kafrelsheikh, Egypt

⁶Department of Animal Physiology, Faculty of Veterinary Medicine, Kafrelsheikh University, Kafrelsheikh, Egypt

*Corresponding Author: Eman Moustafa Moustafa Moustafa, Fish Diseases and Management Department, Faculty of Veterinary Medicine, Kafrelsheikh University, Kafr El-Sheikh Governorate, Egypt. Email: emantarek2002 [at] yahoo.com

Submitted: 22/1/2025 Accepted: 13/3/2025 Published: 31/3/2025

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ABSTRACT

Background: Adding oregano essential oil (OEO) as a supplement has great potential to improve fish production efficiency.

Aim: Current research was executed to assess the prospective impact of OEO incorporation into tilapia diets on physiological response, growth performance, oxidative capacity, and immune status when subjected to Aeromonas hydrophila infection.

Methods: Three different experimental diets were prepared by augmenting a 30% protein basal diet with OEO at concentrations of 0, 0.5, and 1 g/kg feed. Each of the three treatment groups consisted of sixty fish (three copies of twenty fish each) and was randomly assigned out of 180 healthy Nile tilapia (O. niloticus) fingerlings. For 8 weeks, fish in nine separate glass tanks were given individualized diets containing 3% biomass. The survival rate (SR%) of the fish was measured after 15 days of observation following intraperitoneal injection of pathogenic A. hydrophila.

Results: Physiological performance, antioxidant indicators, immunological function (phagocytic activity, lysozyme, and phagocytic index), and growth metrics were all enhanced in fish that were given OEO supplements. Antibiotics may be a new contender: oregano oil, when consumed orally, reduces pathogenic oxidative stress. The villus height, breadth, and depth were enhanced by oregano oil, which also improved intestinal morphology. On the other hand, animals infected with A. hydrophila exhibited signs of deterioration in the spleen, hepatopancreas, and intestinal tissues.

Conclusion: For optimal development, immunity, performance in physiological tests, and gut health, Nile tilapia fingerlings are fed half a gram per kilogram of OEO.

Keywords:Aeromonas hydrophila, Nile tilapia, Oregano essential oil, Oxidative status.

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Introduction

Aquaculture is a key contributor to global nutritional security, addressing the needs of an expanding population (Wang et al., 2018; Heluy et al., 2020). Its rapid growth provides a substantial source of animal protein for humans (Kari et al., 2022). In Egypt, aquaculture is the leading sector for fish production (Moustafa et al., 2024b). However, the stress levels of aquatic animals and their environments have been elevated as a result of the intensification of aquaculture practices to achieve higher yields has increased stress levels in aquatic animals and their environments (Herrera et al., 2019). These pressures have increased disease outbreaks, endangering productivity and sector viability.

Antibiotics and chemical agents, including chlortetracycline, oxytetracycline, co-trimoxazole, sulphadiazine, sulphamethoxazole, and amoxicillin, are often employed for the treatment of diseases in aquaculture (Aliko et al., 2018). However, their extensive use has led to issues, including potential environmental and health hazards. This has driven the urgent need for sustainable alternatives that promote the growth and safeguard the health of aquatic organisms (Dawood et al., 2019b). Beneficial microorganisms from terrestrial sources play critical roles in improving aquatic animal health by boosting immunity, enhancing nutrient absorption, and enhancing resistance to opportunistic pathogens (Yan et al., 2017). Intestinal microbiota function as intended when beneficial bacteria communicate with the host’s digestive system and affect host biological functions (Ramirez and Romero, 2017).

The inclusion of functional feed additives in aquaculture diets provides various benefits (Abdel-Latif et al., 2020b). Common additives include prebiotics, probiotics, medicinal herbs, immunostimulants, trace minerals, and vitamins (Yao et al., 2020). Studies have shown that herbal supplements and extracts enhance growth, antioxidant defenses, and immune function in aquatic animals. Multiple studies have highlighted the ability of herbal extracts to improve immune responses and resilience against environmental stress (Adel et al., 2021; Magouz et al., 2021).

According to Sutili et al. (2018), essential oils exhibit diverse biological functions owing to the natural volatile molecules that plants create. The chemicals carvacrol and thymol, found in abundance in oregano essential oil (OEO) produced from Origanum vulgare L., are recognized for their ability to promote development, fortify the immunological status, reduce inflammation, and kill bacteria (Govaris et al., 2010). Research has demonstrated that common carp (Cyprinus carpio), rainbow trout (Oncorhynchus mykiss), and Nile tilapia (Oreochromis niloticus) benefit economically and physiologically from including OEO in their diets (Abdel-Latif and Khalil, 2014; Haghighi et al., 2018).

Among the many fish species cultivated for human consumption, Nile tilapia (O. niloticus) is second to none in terms of output. Conforming to El-Asely et al. (2020) and Mengistu et al. (2020), among its many admirable qualities are its rapid expansion, low production costs, market demand, and resilience in the face of extreme environmental stress. According to Dawood et al. (2020a), these traits make it an excellent choice for aquaculture systems that involve intense and superintensive practices. However, tilapia are subject to stress factors like overcrowding, low dissolved oxygen, ammonia buildup, and poor water quality when farmed intensively. Injury, disease transmission, and poor health are more likely in such settings (Telli et al., 2014; Ran et al., 2016). This commercially essential species has an impressive tolerance to stress, but it could benefit from techniques that increase its productivity and resistance to infection (Konnert et al., 2022; Dawood et al., 2023).

This investigation was excited to investigate the prospective impact of incorporating OEO into Nile tilapia diets on growth, physiological functions, oxidative balance, and immune function when subjected to Aeromonas hydrophila challenge.

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Materials and Methods

Ethical approval

The ongoing study complied with all relevant regulations and the directives established by Kafrelsheikh University’s Animal Ethics Committee, Egypt (KFS-IACUC/137/2023) on experimental procedures, animal welfare, and protocol.

Preparation of the experimental diet

The following commercial items were utilized to make a regular basal diet: corn gluten meal, fish oil, wheat bran, yellow maize, fish meal, soybean meal, and mineral and vitamin premixes (Table 1). With the use of a feed mill, the dry materials were reduced to fine powder. Three distinct experimental diets were created by adding different quantities of OEO to the basal diet: 0, 0.5, and 1 g/kg. All the ingredients were measured and blended for 20 min. Then, a pelleting machine with a 2–3 mm die size was used to extrude the dough, which had been previously mixed with water, oil, dicalcium phosphate, vitamins, and minerals. After air-drying, the pellets were placed in a freezer. Using the protocols set out by the AOAC (2012), we were able to determine the nutritional profile of the diets.

Experimental Design

We supplied 180 young, healthy Nile tilapia (O. niloticus) fish sourced from a private aquaculture facility in Kafrelsheikh province, Egypt. The fish had an average weight of 16.00 ± 2.00 g when they were first acquired. For 2 weeks, the fish were acclimated to their new environment in a fiberglass tank while eating an essential diet containing 30% crude protein. After acclimation, the fingerlings were accidentally allotted to one of three groups, consisting of sixty fish each. Within each group, 20 fish were replicated. Aeration systems were installed in glass aquariums of 60 × 30 × 40 cm glass aquariums, which held 70 l of water, where fish were placed. One group was provided as a control and received a basal diet. The other two groups received diets supplemented with OEO at concentrations of 0.5 and 1 g/kg, respectively. Feed levels were changed biweekly depending on weight changes for 8 weeks, with fish fed 3% of their total biomass daily. Every day, dechlorinated water was integrated into the aquarium to replenish half of the water, and waste was removed via siphoning.

Using a Lamotte water analysis apparatus (USA), water quality was checked every other week. The temperature was maintained between 24°C and 27°C, dissolved oxygen at 6.5 ± 0.5 mg/l, pH at 7.1 ± 0.8, and ammonia at 0.1 mg/l, and a 12:12-hour light-dark photoperiod was observed, as shown in Table 2. The criteria used to evaluate water quality were outlined by the American Public Health Association APHA (1989).

Table 1. Experimental diet and composition of nutrients of Nile tilapia.

Table 2. Water quality parameters measured in this study.

Assessment of growth parameters

Growth performance was evaluated after 8 weeks. Twenty fish were weighed per replicate using an electronic balance. Growth metrics, including specific growth rate (SGR), total weight gain, average daily gain (ADG), feed conversion ratio (FCR), and survival rate (SR%), were estimated following Moustafa et al. (2024a).

Hematological and biochemical analyses

A day before sampling, the fish were fasted. According to Dawood et al. (2020b), tricaine methane-sulfonate anesthesia at a concentration of 100 mg/l reduced stress. In each group, nine fish were sampled, with three fish in each group. According to Feldman et al. (2000), 5-ml syringes were used to collect blood from the caudal vein. The blood was separated for rapid hematological and biochemical tests. Because of the small quantities, it was necessary to pool the blood samples of the fish (Urbinate and Carneiro, 2006). RBCs were collected using a hemocytometer (Houston, 1990). While Zink’s (1986) method tested (Hb) concentrations, the microhematocrit approach measured (PCV%) (Decie and Lewis, 2006). The methods used to identify MCHC and MCV were based on those of Wintrobe (1934) and Dacie and Lewis (1995). According to Moustafa et al. (2024a), the white blood cell counts were separated. The method outlined by Moustafa et al. (2024b) was used to measure albumin, total protein, globulin, serum AST, ALT, urea, and creatinine.

Immune Response parameters

We measured phagocytic activity (PA) and its index using methods described by Kawahara et al. (1991) and Magouz et al. (2019). Demers and Bayne (1997) found that lysozyme activity could be determined by lysing Micrococcus lysodeikticus (Sigma, St. Louis, MO). The results of the blood glucose and cholesterol tests were determined by colorimetry according to the methods developed by Caraway and Watts (1987) and Richmond (1973). Olmos and Henderson (1999), Caraway (1959), and Fassati and Prencipe (1982) used colorimetry to analyze triglycerides, lipase, and amylase. In their evaluation of antioxidant markers, Moustafa et al. (2024b) considered GPx, MDA, SOD, and CAT.

Challenge with A. hydrophila

The fish were given an I/P injection of 0.2 ml of a bacterial suspension containing 3 × 107 CFU in order to determine their resistance to Aeromonas hydrophila (Li et al., 2011). The mortality rate was tracked every day for 15 days after infection. After cultivating the bacteria on TSA plates, the drop-plate method was used to determine the colony-forming units (Cruickshank, 1975). In accordance with Reed and Muench (1938), the LD50 dose was determined.

Gene expression analysis

Trizol was used for RNA extraction from 50 mg of spleen tissue from O. niloticus. Then, agarose gel electrophoresis and a spectrophotometer were used to ensure that the RNA was of good quality and concentration. Gene expression profiling was conducted using gene-specific primer sequences in Rotor Gene-Q (Qiagen-Germany) to amplify inflammatory (immunity)-related genes such as Nf-κb. https://www.ncbi.nlm.nih.gov/gene/4790TNF-α, IL-1β. Antioxidant-related genes as CAT and GPx (Table 3). A synthesis kit was used to reverse-transcribe two micrograms of RNA into cDNA. Utilizing real-time PCR with particular primers, the gene expression of antioxidant and immunological indicators, such as GPx, Nf-κb, TNF-α, IL-1β, and CAT, was checked. The 2-ΔŔCT technique, following Livak and Schmittgen (2001), was used to standardize the results to the housekeeping genes β-actin and EF1A.

Table 3. Primers sequences used for qRT–PCR analysis.

Histopathological examinations (Morphometry of intestinal villi)

There were five fish in the control and oregano oil treatment groups. For intestinal histomorphometry, we randomly selected 0.5 and 1 gm/kg. Samples of the anterior intestine were taken during dissection of the abdomen performed under anesthesia with 40% ethyl alcohol. After 18–24 hours in Bouin’s solution, tissue samples were dehydrated by adding increasing percentages of ethyl alcohol, ranging from 70% to absolute alcohol. Paraffin wax and xylene were used to clean the dried materials. According to Bancroft and Gamble (2007), hematoxylin and eosin were used to stain 4–5 μm slices for histological and morphological examination. Intestinal villi dimensions were determined using a method developed by the National Institutes of Health. Ten villi and crypts linked to the villus were randomly selected from five intestinal cross-sections to determine the average ( ± SE).

Statistical analysis

The data were expressed as mean ± SEM, were investigated utilizing GraphPad Prism (v9.5, GraphPad Software, USA). One-way ANOVA was conducted, followed by Tukey’s multiple comparison test was administered (p < 0.05).

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Results

Growth performance

The effects of OEO on the development of O. niloticus were investigated in this study. The results are presented in Table 4, which shows that all groups experienced substantial improvements in final body weight, feed intake, weight increase, ADG, and final fish biomass. The T2 group exhibited the most significant values associated with the control treatment (T1) (p < 0.05). Among the treated treatments, the FCR was meaningfully decreased in the T2 group compared with the other groups. No matter which group you looked at, the survival rate (SR%) was always 100%.

Hematological parameters

Each group that received OEO had markedly elevated levels of RBC, Hb, PCV, and MCV than the control (T1) treatment (p < 0.05). The treatment groups did not differ significantly, though (p > 0.05). There were no discernible differences between the experimental and control groups (p > 0.05) in terms of MCHC or MCH (Table 5). The differential leukocyte count (WBCs) and lymphocytes significantly increased (p < 0.05) in the treated groups compared with the control, with the T3 group exhibiting the highest values. Basophils, eosinophils, monocytes, and heterophils showed no significant variations in any groups and control (p > 0.05) (Table 6).

Biochemical analysis

Table 7 shows that oregano treatment significantly decreased AST and ALT levels (p < 0.05) across all groups compared with the control group. Albumin, total protein, and globulin levels increased dramatically in the T2 group (p < 0.05). Compared with the control group, all oregano-treated groups had considerably lower creatinine levels, whereas the T3 group had significantly higher urea levels (p < 0.05). The treatment groups also showed markedly reduced levels of glucose, triglycerides, and cholesterol compared with the control group.

Table 4. The effect of Oregano essential oil on the growth performance of Oreochromis niloticus.

Table 5. Effect of Oregano essential oil on the hematological parameters of Oreochromis niloticus.

Table 6. Effect of Oregano essential oil on the leukocytic counts of Oreochromis niloticus.

Table 7. Effect of Oregano essential oil on the biochemical parameters of Oreochromis niloticus.

Table 8. Effect of Oregano essential oil on the immunity of Oreochromis niloticus.

Table 9. Effect of Oregano essential oil on the immunity of Oreochromis niloticus to A. hydrophila.

Immunological and antioxidant parameters

In comparison with the control group, the treated groups showed a substantial improvement (p < 0.05) in immunological indicators, such as phagocytic index, PA, and lysozyme activity. The T2 group had the highest values for these parameters (Table 8). The treatment groups also demonstrated substantial upregulation of amylase and lipase activity, with T2 exhibiting the highest levels (p < 0.05).

The antioxidant analysis demonstrated that oregano treatment markedly elevated CAT, SOD, and GPx levels relative to the control treatment (p < 0.05). T3 had the highest CAT and GPx values. All groups treated with oregano had lower MDA activity, indicating reduced lipid peroxidation (Table 8).

Immunological and antioxidant parameters after A. hydrophila challenge

Concerning the immunological and antioxidative measures after challenge of Nile tilapia with Aeromonas hydrophila; phagocytic index, activity, lysozyme activity, amylase, and lipase levels were substantially elevated (p < 0.05) in all treatment groups compared with the control treatment after the A. hydrophila challenge. As indicated in Table 9, T2 had the highest values. The treated groups had considerably more significant antioxidant activity levels of (CAT, SOD and GPx) relative to the control group (p < 0.05). CAT and GPx were most significant in T2 and T3. Oregano significantly reduced MDA activity after treatment.

Relative gene expression

For the preinfection group, relative gene expression levels of inflammation-related cytokines and antioxidant-related genes were assessed in the spleen of Nile tilapia across different experimental groups (Fig. 1). Regarding inflammation-related genes, NF-κB, there were no discernible disparities in the relative gene expression between control, oregano 0.5, and oregano 1. In the case of TNF-α and IL-1β, no substantial changes were investigated between oregano 0.5 and oregano 1, but both showed higher expression levels related to the control. For antioxidant genes, CAT and GPX levels were both higher in the oregano 1 group, followed by oregano 0.5, and both were elevated relative to the control.

Fig. 1. Differential expression of inflammation-related genes and antioxidant-related genes in the spleen of Nile tilapia groups fed on oregano 0.5 and oregano 1. (A) Nf-κb: Nuclear factor kappa B, (B) TNF-α: Tumor necrosis factor alpha, (C) IL-1β: Interleukin 1β, (D) CAT: Catalase, and (E) GPx: glutathione peroxidase. Columns with different superscript letters in the same figure are significantly different (p ≤ 0.05) are significantly different (p ≤ 0.05).

Conversely, following A. hydrophila infection, the gene expression levels of antioxidant-related genes and inflammation-related cytokines were evaluated in Nile tilapia spleens from different experimental groups (Fig. 2). In terms of inflammation-related genes, NF-κB expression was elevated in the oregano 0.5 group relative to oregano 1, with both levels elevated above the control. Regarding TNF-α, no substantial differences were detected between the oregano 0.5 and oregano 1 groups, but both groups exhibited higher TNF-expression than the control. For IL-1β, the highest gene expression was observed in the oregano 0.5 group, followed by oregano 1, with both levels higher than the control. Regarding antioxidant genes, CAT and GPX expression levels were highest in the oregano 1 group, followed by oregano 0.5, and both were significantly elevated compared with the control.

Fig. 2. Differential expression of inflammation-related genes and antioxidant-related genes in the spleen of Nile tilapia groups fed on oregano 0.5 and oregano 1. (A) Nf-κb: Nuclear factor kappa B, (B) TNF-α: Tumor necrosis factor alpha, (C) IL-1β: Interleukin 1β, (D) CAT: Catalase, and (E) GPx: glutathione peroxidase. Columns with different superscript letters in the same figure are significantly different (p ≤ 0.05) are significantly different (p ≤ 0.05).

Histopathological findings

Histological analysis of the intestinal wall revealed a tunica mucosa, including numerous villi lined with columnar epithelium and goblet cells, supported by the lamina propria. The treated groups exhibited denser, longer, and more branched intestinal villi than the control group, indicating enhanced intestinal morphology (Table 10 and Fig. 3).

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Discussion

Dietary supplementation in aquaculture can enhance yield and reduce mortality induced by abiotic and biotic stressors (Wee et al., 2022; Yousuf et al., 2022). The aquafers industry’s primary goal is to attain two goals: enhanced development and resilience in the majority of cultured fish species, together with preventive healthcare through utilizing diverse healthy methodologies to ensure the feed’s controllability within the aquaculture system (Kiron, 2012). The immune status of cultivated fish species is predominantly influenced by their dietary state, which is essential for achieving a higher level of confidence (Dawood et al., 2018).

Table 10. Intestinal Morphometric analysis of O. niloticus-treated groups fed diets containing various concentrations of Oregano essential oil.

Fig. 3. H&E-stained histomicrograph of the anterior part of the intestine of O. niloticus of control (A), oregano essential oil 0.5 g (B) and oregano essential oil 1 g (C) showing intestinal villi (arrow heads), lamina epithelialis (E), lamina propria (P), and lamina muscularis (M).

A useful food supplement is a crucial management device for enhancing various growth parameters and resistance to diseases in aquacultured fish (Bharathi et al., 2019). In aquaculture, the use of antibiotics has raised public concern because of the heightened potential for the development of antibiotic-resistant bacteria in ecosystems. This poses a danger to ecosystems, aquaculture, consumers, and terrestrial animals (Ghiasi et al., 2021). However, functional additives offer a safe and healthy option for aquaculture (Abdel-Latif and Khalil, 2014; Dawood et al., 2020). Phytobiotics, a class of naturally occurring additives, are extensively employed in fish medicine (Abdel-Tawwab, 2016; Jana et al., 2018).

Growth performance

The results showed that all groups treated with OEO showed improvement in growth parameters, whereas the T2 group (given 0.5 g/kg OEO) showed the greatest improvement. OEO probably boosted the utilization of feed, which is why these findings occurred. Past research on the influence of OEO on different fish species, including rainbow trout, Nile tilapia, common carp, and gilthead seabream, has shown similar outcomes (Haghighi and Rohani, 2015; Pourmoghim et al., 2015; García-Beltrán et al., 2018, 2020; Shourbela et al., 2021; Roldan-Juarez et al., 2023). One reason OEO promotes growth is its ability to remain effective even in the stomach’s acidic environment. Its aromatic components also make feed more appetizing, which may affect genes in the pituitary gland that control hunger (Aydin and Barbas, 2020). Also, production costs were reduced due to improved feed conversion rates, which meant less feed was needed for each unit of fish growth (Roldan-Juarez et al., 2023).

Hematological parameters

All groups fed oregano had considerably more significant levels of RBCs, Hb, PCV, and MCV, with the T2 group showing the most improvement in blood biochemical tests, which are frequently used to monitor fish health when functional feed additives are administered (Dawood et al., 2019a). The treatment groups also had higher white blood cell and lymphocyte counts than the control groups, suggesting that the immune system was working better after taking OEO. Similar conclusions were reached by Mohammadi et al. (2020) and Shourbela et al. (2021). Hematological markers offer essential information on general well-being, immunity, and resistance to disease (Fazio, 2019; Magouz et al., 2020).

Biochemical analysis

The groups that received oregano supplements had higher levels of biochemical indicators that are crucial for assessing metabolic and immunological functioning, including albumin, total serum protein, and globulin (Yilmaz, 2020). The results are consistent with previous findings in rainbow trout (Pourmoghim et al., 2015) and common carp (Abdel-Latif et al., 2020b). According to Zhang et al. (2020), the carvacrol and thymol found in OEO are renowned for their ability to reduce stress and enhance the immune status. In addition to decreased creatinine and increased urea, the oregano-fed groups exhibited elevated ALT and AST levels.

Due to thymol’s regulating effects on liver and kidney function (Zhang et al., 2020), comparable trends were detected in rainbow trout (Hoseini and Yousefi, 2019). Elevated levels of AST and ALT (liver function markers), together with heightened urea and decreased creatinine (renal function markers) levels, were observed in the OEO-supplemented groups. However, these findings differ from those of Abdel-Latif et al. (2020b), who reported decreased levels of these markers in common carp. The stress-relieving effects of OEO are supported by lower glucose levels in the treated groups, which aligns with the findings of Dawood et al. (2020a), who indicated that tilapia raised at high densities exhibited elevated glucose levels; however, dietary β-glucan mitigated these levels.

Immunological and anti-oxidant parameters

The T2 group, in particular, showed improved phagocytic, lysozyme, phagocytic index, amylase, and lipase activities, indicating enhanced immunological responses. The bioactive ingredients thymol and carvacrol, the principal constituents of OEOs, may be responsible for these effects (Sivropoulou et al., 1996). In accordance with these findings, other investigations have shown the substantial impact of dietary OEOs on the augmentation of fish non-specific immunity (Zheng et al., 2009; Mabrok and Wahdan, 2018).

This includes rainbow trout (Yilmaz et al., 2015) and Nile tilapia (Ran et al., 2016; Abdel-Latif et al., 2020a). However, when channel catfish were fed diets containing only carvacrol or thymol, Zheng et al. (2009) found no discernible changes in serum lysozyme activity.

Concerning the antioxidative parameters, the results showed that those administered oregano had better antioxidant status, with lower MDA levels and higher SOD and CAT activity. Carvacrol and thymol’s inherent scavenging and chelating capabilities are responsible for these antioxidant actions (Abdel-Latif et al., 2020b). There is agreement between these results and previous studies on common carp (Abdel-Latif et al., 2020b), rainbow trout (Giannenas et al., 2012; Diler et al., 2017; Rafieepour et al., 2019), Koi carp (Zhang et al., 2020), and Nile tilapia (Abdel-Latif and Khalil, 2014). On the other hand, variations in species, research duration, or oregano formulations led García-Beltrán et al. (2020) to conclude that oregano leaf powder exerted no substantial influence on the antioxidant activities of gilthead seabream. Sahin et al. (2014) determined that antioxidants combined with essential oils serve as advantageous supplements to mitigate immunological deficiencies resulting from oxidative stress, as well as to enhance development and stress responses linked to intense cultivation conditions.

Immunological and antioxidant parameters after A. hydrophila challenge

All immunological, amylase, and lipase activities were substantially upregulated, especially in the T2 group treated with OEO and subjected to A. hydrophila challenge. Antioxidant activities were higher in all augmented challenged treatments than in the control treatment. The outcomes coincided with former studies; Nile tilapia resistance to pathogenic Vibrio alginolyticus (Abdel-Latif and Khalil, 2014); pathogenic Edwardseilla tarda (Rattanachaikunsopon and Phumkhachorn, 2010); pathogenic A. hydrophila (Seden et al., 2009); Koi carp resistance to A. hydrophila (Zhang et al., 2020); and channel catfish resistance against A. hydrophila (Zheng et al., 2009).

The antibacterial activities of carvacrol and thymol are responsible for the increased protection against Aeromonas hydrophila in Nile tilapia fed oregano. These chemicals cause bacterial membrane disruption, which in turn changes cellular integrity, ionic homeostasis, and pH balance (Lambert et al., 2001; Abdel-Latif et al., 2020b). In addition, groups treated with oregano showed increased production of genes linked to the immune status, such as TNF-α and IL-1β, which confirms that the immune system was more prepared and inflammation was reduced. Research on the utilization of essential oils and probiotics in fish feeding has shown similar results (Dawood et al., 2022).

Relative gene expression

The oxidative, physiological status, and gene expression levels of SOD, GSH-Px, IL-1β, and TNF-α, are all associated with the health of the fish immune system (Morano, 2007; Elbahnaswy and Elshopakey, 2020). Groups treated with oregano showed increased production of genes associated with the immune system, such as cytokine tumor necrotic factor-alpha (TNF-α) and pro-inflammatory interleukin 1β (IL-1β), confirming that the immune system was more prepared and inflammation was reduced. Research on the use of essential oils and probiotics in fish nutrition has shown similar results (Dawood et al., 2022).

Histopathological findings

Oregano supplementation significantly improves gastrointestinal health, which is critical for nutritional absorption and digestion. The supplement increased villus height, width, and crypt depth. Previous research by Abdel-Latif et al. (2020b), Xin et al. (2022), and Roldan-Juarez et al. (2023) found that an OEO derived from tilapia improved intestinal morphology. Our findings are in line with theirs. Improved health, improved digestion, and efficient feed utilization are all outcomes of a fish with a well-structured digestive system (Gao et al., 2013).

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Conclusion

This study expands our understanding of the practical dietary benefits of OEO in fish diets. This study showed that Oreochromis niloticus fingerlings fed OEO had better growth rates, physiological responses, and pathogen resistance; all of which are important in modern intensive aquaculture. OEO, when incorporated in Nile tilapia diets, effectively reduces the stress caused by Aeromonas hydrophila and improves immunological markers in fish exposed to pathogens, making it a viable alternative to antibiotics. Because of the increased rate of growth, OEO (0.5 g/kg) should be added to fingerling Nile tilapia diets.

Conflict of interest

No conflicts of interest.

Author contributions

All authors contributed to the study and manuscript. Eman Moustafa and Mohamed Khallaf planned the study. Wesam Marzouk, Mohamed Khallaf, Hanan Ghetas, Haguer Salah El Din, and Eman Moustafa handled data collection and analysis. Azza Hafez prepared the fish diet, Wesam Marzouk conducted the statistical analysis, Mona Assas performed the gene expression analysis, and Alaa Elgaabari conducted the blood examination. Early drafts were written by several authors, with Eman Moustafa revising the final manuscript. All authors have reviewed and approved the final version.

Funding

This study did not use external financial sources.

Availability of data

Upon request.

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