Using some natural essential oils and their nano-emulsions for ammonia management, anti-stress and prevention of streptococcosis in Nile tilapia, Oreochromis niloticus

The current study aimed to investigate the efficacy of dietary thyme essential oil (TEO), Nigella sativa essential oil (NSEO), thyme essential oil nano-emulsion (TEO-NE), and Nigella sativa essential oil nano-emulsion (NSEO-NE) in reducing total ammonia nitrogen (TAN), improving immune response, mitigating stress, and acting as anti-inflammatory agents as well as preventing streptococcosis infection in Oreochromis niloticus (O. niloticus). Fish (N = 330, 14 ± 2 g) were divided into 10 groups of 11 fish each, with three replicates in each group. The negative and positive controls were fed a control diet, while the third group was given 1% TEO. Moreover, the fourth group of fish was given 2% TEO. The fifth and sixth groups were fed 1% TEO-NE and 2% TEO-NE, respectively. The seventh, eighth, ninth, and tenth groups were fed 1% NSEO, 2% NSEO, 1% NSEO-NE, and 2% NSEO-NE, respectively. After dietary intake of TEO, NSEO, and their nano-emulsions for 28 days, the mean values of TAN levels in the water of fish aquaria had a significant reduction in the group fed 2% TEO-NE compared to the control group. On the contrary, NSEO-NE at the same concentration had no significant effect on TAN levels. The levels of lysozyme, complement 5, and IgM increased in all feeding groups compared to the control group. Concerning cortisol level as a stress indicator, it was decreased in all feeding groups compared to the control. Also, the current experiment overall showed a significant decrease in the expression level of pro-inflammatory tumor necrosis factor (TNF-α) gene in the gills of fish groups fed TEO, TEO-NE, NS, and NS-NE relative to the β-actin gene. Oppositely, there was an increase in the expression level of the anti-inflammatory transforming growth factor (TGF-β). In the current study, TEO-NE and NSEO-NE showed a better effect on preventing streptococcosis in O. niloticus with no mortality than 1% TEO and NSEO, respectively. Furthermore, there was a 12.5% mortality rate and an 84.99 RPS in the group fed 1% TEO and injected with Streptococcus inae. On the other hand, the groups fed 1 and 2% NSEO showed 37.5 and 25% mortality rate, respectively and 54.99 and 69.99 RPS. In conclusion, the nano-emulsion either TEO or NSEO had the superior effect. For bulk status, the TEO had superior effect than NSEO. The study needs more investigations for ammonia, either on the mode of action or over a longer period.


Introduction
Indeed, aquaculture is one of the fastest-growing food-producing sectors, accounting for one-third of worldwide fisheries production (Rocha, 2022). Aquaculture can benefit from nanotechnologies in various ways (Huang et al. 2015). It seeks to increase the efficacy of materials by altering their size and allowing the nanoscale to have a more significant impact on biology and medicine (Sahlan et al. 2017).
Nile tilapia, Oreochromis niloticus (O. niloticus) is the most widely cultured species among a diverse group of aquatic habitats (FAO 2018) because of its rapid development, stress tolerance, and reproduction in both freshwater and saltwater conditions (Gibtan et al. 2008). Water quality management is the most significant component in tilapia farming, and ammonia content is the most critical water quality factor (El-Gendy et al. 2015). Although tilapia intensification at high stocking densities has resulted in significant productivity gains, it also increases ammonia stress (Atle et al. 2009) and the incidence of infectious diseases, particularly those caused by bacterial pathogens ((FAO 2009 andPękala-Safińska 2018). Furthermore, one of the most common bacterial diseases that cause major economic losses in tilapia production sector is streptococcosis (Gültepe et al. 2014). Streptococcus inae (S. inae) the most common cause of streptococcosis is mostly managed by antibiotics (Gültepe et al. 2014). Since the oral administration of such antibiotics in aqua-feed is prohibited in some countries (Romero et al. 2012), the search for novel natural feed additives to replace antibiotics has become an important and ongoing task (Talbot et al. 1994, Santacruz-Reyes et al. 2012Li et al. 2014;Engler et al. 2018;Guo et al. 2018). Among those selected natural feed additives are natural essential oils which are categorized as ''generally recognized as safe'' (GRAS) and are being exploited as natural antimicrobial additives to replace synthetic antimicrobial compounds (Lv et al. 2011). Similarly, thyme essential oil (TEO) and Nigella sativa essentials oil (NSEO) and their nano-emulsions have a high potential for use in aquaculture systems due to a variety of beneficial biological effects reported in aquatic species, such as reducing total ammonia excretion in sea bass and rainbow trout water (Yılmaz et al. 2012;Ozogul et al. 2017). Also, they have immunomodulatory, anti-stress, anti-inf lammatory (Mbarek et al. 2007), and antibacterial activities against Gram-positive and negative bacteria (Kooti et al. 2016;Aziz et al. 2017;El-Aarag et al. 2017;Kumari et al 2018;Salam et al. 2021). Therefore, the current study was designed to investigate the efficacy of dietary supplementation with TEO and NSEO and their nano-emulsions in reducing ammonia excretion, improving immune response, mitigating stress, and acting as anti-inf lammatory agents in O. niloticus. Also, the prevention of streptococcosis using these natural products was carried out.

Ethical committee
The Faculty of Veterinary Medicine at Beni-Suef University in Egypt's Institutional Animal Care and Use Committee (number: 022-355) approved all experiments.

Collection of experimental fish
For the experimental studies, 438 healthy O. niloticus with 14±2 g average body weight were gathered alive from the fish hatchery of Abo-Saleh in Beni-Suef, Egypt. The captured fish were taken to the Fish Diseases and Management wet laboratory at Beni-Suef University's Faculty of Veterinary Medicine in Egypt. Three fiberglass tanks of 500 L capacity each were filled with fish and supplied with tap water without chlorine and consistent aeration. The experimental fish were acclimatized in these tanks for 14 days and fed a 3% of their body weight of fish ration (Table 1).

Management of experimental fish
After acclimation, fish have been added in glass aquaria of 90×25×40 cm with a water capacity of thirty liters. Through an air blower, each tank received continuous aeration. 1 3 The water rate of exchange in the experimental aquaria was 10% per day. Food was given to the fish at a rate of 3% of body weight. During the trial, water quality was measured twice a week. Water temperature (26 ±1°C) was measured with a water thermometer, dissolved oxygen (D.O; 7 ± 2 mg/L) was measured by a D.O meter (Yellow Spring Instrument Co., USA), and pH (7-8) was determined by pH indicator paper (Fisher Scientific, Denver, CO, USA).

Source of TEO and NSEO
Thyme essential oil (TEO) was purchased from Sigma-Aldrich provided TEO (Chemie GmbH, Steinheim, Germany, containing a pamphlet, supplemented with the oil structure details). However, NS-EO was obtained from a local shop.

Synthesis of TEO-NE and NSEO-NE
Nano-emulsions of TEO and NSEO were prepared according to Pongsumpun et al. (2019) using the ultra-sonication technique. The nano-emulsions were prepared through two stages: the aqueous one, which was prepared by combining tween 80 at a concentration of 3% in distilled water, and the oil phase, which was made up of essential oils diluted to 1.0 and 2 mL/100mL. Then, a coarse emulsion was created by gradually adding the oil phase to the aqueous phase (W/W) and vigorously mixing by a magnetic stirrer (MSH-20D, Wise Stir) at 500 rpm and 25°C for 15 min. An ultrasonic bath was used to combine the two phases, and the result was a nano-emulsion (Ultrasons, P-Selecta). For sonication, a fixed frequency of 43 kHz, output at 210 W, and high levels of oscillation were used. The ultrasound was generated and exposed to a water bath at 25 °C for 10 minutes.

Characterizations of TEO-NE and NSEO-NE
Transmission electron microscopy (TEM) was used to characterize TEO-NE and NSEO-NE. For the TEM procedure, 20 microliters of diluted samples were placed for 10 min on a film-coated 200-mesh copper specimen grid. Next, one drop of 3% phosphotungstic acid was used to stain the grid, and it was left for three minutes to dry. After drying, the coated grid was examined with a TEM microscope (Philips, CM 12). Operating at 120 kV allowed for the observation of the samples. Faculty of Agriculture Central Laboratory at Cairo University in Egypt carried out this work. Also, the samples were characterized by zeta potential at the Faculty of Postgraduate Studies of Advanced Science, Beni-Suef University, Egypt. Zeta potential was operated by adding the emulsion sample (25 ul) into a capillary cell and diluted in 2 ml water. The emulsion was conditioned for 20 minutes before use (Sze et al. 2003).

Formation of diet
The fish ration was powdered into an extremely fine dust using a mortar and pestle. The TEO and NSEO, as well as their nano-emulsion, were combined with the previously made fine powder to produce five fish diets. There were no additives in diet 1 (control), 1% TEO in diet 2, 2% TEO in diet 3, 1% TEO-NE in diet 4, and 2% TEO-NE in diet 5. Diets 6 and 7 each contain 1% NSEO and 2% NSEO and diets 8 and 9 each contain 1% NSEO-NE and 2% NSEO-NE (Salam et al 2021). The components of the fish diet were combined with distilled water to create a homogeneous mixture. To create extruded strings that were dried at room temperature; the mixture was run through a manually operated meat processing machine Rattanachaikunsopon and Phumkhachorn (2010).

Design of experiment and regime of feeding
Three hundred and thirty O. niloticus had been separated into 10 groups of 11 fish each and had three replicates to each group. The negative and positive controls were fed a control diet with no additives, while the third group received 1% TEO. Furthermore, the fish in the fourth group were given 2% TEO. The fifth and sixth groups were fed 1% TEO-NE and 2% TEO-NE. The seventh, eighth, ninth, and tenth groups were fed 1% NSEO, 2% NSEO, 1% NSEO-NE, and 2% NSEO-NE, respectively. During the experiment, each group received 3% of their body weight of their designated food divided into two times a day for 28 days. Total ammonia nitrogen (TAN) was monitored throughout the experiment. Three fish from each group were used to collect serum, plasma, and gills at the end of the feeding period. For the purpose of preventing streptococcosis, the remaining eight fish in each group with their replicates were used.

Measuring TAN in the fish glass aquaria
For four weeks, water samples were collected twice per week. In sterile, colorless glass Stoppard bottles with 1 L capacities, water samples were taken separately from each group of glass aquariums at a depth of 10 cm. To the Department of Hygiene, Zoonoses and Epidemiology, Faculty of Veterinary Medicine, Beni-Suef University, Beni-Suef, the labelled bottles were shipped at 5°C. With the aid of a Multiparameter Photometer, TAN levels were determined (bench, HI83200, HANNA, Romania). The concentration was measured using the Nessler method and reagent kits HI93700A-0 and HI93700B-0. The TAN level was also calculated in parts per million (ppm).

Collection of serum, plasma and gills of experimental feeding groups
Three fish from each group were netted and anesthetized by tricaine methane sulfonate (Sigma-Aldrich Chemical Co., Cairo, Egypt) after 28 days of feeding to collect their blood and gills. Serum was prepared by collection of blood from caudal veins without anticoagulant while the remaining blood was drawn and put into cooled plastic tubes having 3 mg of Na2EDTA for plasma collection.
To estimate the pro-inflammatory tumor necrosis factor (TNF--α) and the anti-inflammatory transforming growth factor (TGF-β) genes, the collected pieces of gills were placed in tubes of 2 ml containing RNAlater solution (Merc, Egypt). Following an overnight period at 4 °C in the refrigerator, the samples were then frozen at -80 °C.

Estimation of immune parameters and stress indicators in serum and plasma of feeding groups
Lysozyme, complement 5, and IgM concentrations in the collected serum were determined using test kits from CUSABIO (Fish Lysozyme (LZM) ELISA Kit Catalog Number, CSB-E17296Fh, China), Fish Complement 5 (C5) ELISA Kit Catalog Number, CSB-F13502F, China), and Fish immunoglobulin M ELISA Kit Catalog Number, CSB-E12045Fh, China). Additionally, the plasma cortisol level was measured using a cortisol ELISA kit® (Calbiotech, catalogue No. CO103S, Canada) following manufacturer's instructions, and according to Schlaghecke et al. (1992) results were calculated using an automatic ELISA reader (SUNRISE®; Tecan, Austria).

Protocol of expression of pro-inflammatory TNF-α and anti-inflammatory TGF-β genes in the gills of the experimental feeding groups
Total RNA was isolated using TransZol Reagent (Transgen Biotech, Beijing, China) following the manufacturer's instructions. The RNA concentration (OD 260nm) and purity (OD 260nm/OD280 nm ratio, range 1.90-2.08) of each sample was measured using Nan-oDrop2000c (Thermo Scientific, Massachusetts, USA). The integrity of RNA was checked by 1.0% agarose gel electrophoresis. One microgram of total RNA from each sample (4 samples from 10GCI group and 5 samples from every other group) was reverse transcribed to obtain cDNA using Prime Script™RT reagent Kit with gDNA Eraser (TaKaRa, Dalian, China) to remove genomic DNA contamination, following the manufacturer's protocol. Expressions of different immune-related genes were studied by real time PCR. The primers used were listed in Table 2, using β-actin as the reference gene. SYBR green reagent kit used in the real-time PCR experiments was KAPA SYBR® qPCR Kit Master Mix (2X) Universal (KAPA Biosystems, California, USA). Each sample was separately treated in triplicate in a 10μl final volume, including 200nM of each primer ( Table 2) and 10ng of cDNA template. PCR amplification cycles were performed using Roche LightCycler®480 system (Roche, Shanghai, China). The cycling profile was as follows: enzyme activation was carried out at 95°C for 3min, followed by 40 cycles of denaturing at 95°C for 3s, and annealing at 60°C for 30s. Results of real-time PCR were analyzed using 2-ΔΔCt method (Livak et al. 2001). The transcript abundance of each target gene was obtained as Ct value, and normalized by that of β-actin gene as an internal reference. The relative expressions of genes were expressed as mean ± SE, and ANOVA with Dunnett test was used to compare the expression differences between experiment groups and control group in IBM SPSS Statistics 22.0 ( Table 2).

Determination of the median lethal dose (LD50) of S. inae in O. niloticus
For median lethal dose (LD 50 ) determination of S. inae in O. niloticus, about 108 apparently healthy O. niloticus were divided into 6 groups (6 fish per group) with three replicates. An overnight culture of S. inae was prepared at densities of 1.5 × 10 8 , 1.5 × 10 7 , 1.5 × 10 6 , 1.5 × 10 5 and 1.5 × 10 4 CFU/mL. Each dilution was injected intraperitoneally into a fish group at a dose of 200 μL/fish and the 6 th group was injected with 200 μL of physiological saline (control negative). All fish groups were kept for 2 weeks, and mortalities were recorded daily.

Prevention of streptococcosis in O. niloticus
The S. inae BNS 0014 strain was kindly obtained from the department of Fish Diseases and Management, Faculty of Veterinary Medicine, Beni-Suef University, Beni-Suef, Egypt. The strain was isolated from diseased O. niloticus (Hussein and Hassan 2011).
After 28 days of feeding, the final eight fish from each group, along with their replicas, were injected. The negative control and its replicates were injected with 200 µL physiological saline intraperitoneally. S. inae was administered intraperitoneally to fish in the second (positive control), third, fourth, fifth, sixth, seventh, eighth, ninth, and tenth groups at doses of 200 µL of 1.5×10 7 CFU/mL each. The injected groups were kept in glass aquaria for two weeks (Schlaghecke et al. 1992). The mortality and relative percent survival (RPS) was calculated by the formula proposed by Amend (1981): RPS = 1 (% of mortality in treated groups /% of mortality in control group)×100.

Statistical analyses
All data were assembled and subjected to statistical analysis using the Advanced Models 16.0 software's one-way ANOVA (post hoc test; Dunnet's test) (SPSS, Tokyo, Japan). Statistical significance was defined as p-value < 0.05 was considered statistically significant.

Characterization of TEO-NE and NSEO-NE
The TEM photography of the TEO-NE revealed that it had a spherical shape between 72.4 and 58.9 nm (Fig. 1). TEO-zeta NE's potential ranged from 606.1 to 789.0 nm (Fig. 2). The droplets in the nano-emulsion appeared dark; however, the TEM micrograph of NSEO-NE shows the actual size and shape. The TEM photography revealed that the NSEO-NE was spherical and measured between 16.4 and 13.3 nm in size (Fig. 3). NSEO-zeta NE's potential ranged from 324.7 to 574.0 nm (Fig. 4).

Total ammonia nitrogen (TAN) in the fish glass aquaria during feeding period
The effectiveness of dietary intake of thyme oil, Nigella oil, and their nano-emulsion on mitigation of TAN level in different fish groups was determined during the current trial. Adding TEO at a concentration of 2.0% led to slightly alleviate the mean values of TAN during the trial periods at p ≤ 0.05 whereas the TAN level in the 4 th week of trial was 0.41±0.0 mg/L compared to control one and pre-experiment level ( Table 3.

Immune parameters and stress indicators in serum and plasma of experimental groups
Lysozyme, complement 5, and IgM levels increased in all feeding groups following the administration of various concentrations of TEO, TEO-NE, NSEO, and NSEO-NE compared to control group. Compared to the control, all feeding groups had lower cortisol levels (Table 4).

Expression of pro-inflammatory TNF-α genes and anti-inflammatory TGF-β in gills of feeding groups
The current experiment overall showed a significant decrease in expression of pro-inflammatory TNF-α gene in fish gills fed TEO, TEO-NE, NS and NS-NE relative to the β-actin gene. Oppositely, there was increase in expression level of the anti-inflammatory TGF-β gene in fish fed TEO, TEO-NE, NS and NS-NE relative to the β-actin gene ( Table 5).

The LD 50 of S. inae in healthy O. niloticus
The majority of fish mortality occurred during the first week and the LD 50 was 1.5×10 5 CFU/ml (Fig. 5).

Streptococcosis prevention in O. niloticus using TEO and NS and their nano-emulsion
The mortality rate was zero but RPS was 100% in groups fed a high concentration of TEO, as well as high and low concentrations of TEO-NE and NSEO-NE, and then injected with S. inae strains, and control negative group that fed a normal diet and then injected with saline. The group given low concentrations of TEO and subsequently injected with

Discussion
Improving fish feed is essential for developing efficient and profitable aquaculture production because it is the most expensive component of the aquaculture industry (Ghanawi et al. 2011). Ammonia is the most harmful aquaculture fish product. It impedes aquaculture intensification and, as a result, fish production El-Gendy et al. (2015) and Tumwesigye et al. (2022). The efficacy of dietary intake of TEO, NSEO, and their nano-emulsion on mitigating TAN levels in different fish groups clarified that a dietary supplement of TEO and its nano-emulsion (TEO-NE) at a concentration of 2.0% had a significant reduction in the mean values of TAN during the 3 rd and 4 th weeks of the trial compared to first two weeks and pre-experiment. Yılmaz et al. (2012) found a slight decrease in the total amount of ammonia nitrogen excretion in the water of sea bass when adding TEO to fish feed. Parallel findings were also obtained using Yucca schidigera as a feed additive in Nile tilapia There is a lack of information in this direction. Those results agreed with the aforementioned studies that specified that a thyme extract significantly reduced the concentration of ammonia in rabbits compared to a control one (Kandeil et al. and Abd El-Azeem et al 2019). On the opposing, NSEO had no significant effect on TAN levels at the highest concentration of 2.0 % during the feeding period of the current trial. Meanwhile, adding NSEO-NE to fish feed at the same concentration (2.0%) exhibited significant mitigation in TAN levels especially during the 3 rd and 4 th week of the trial. Ozogul (2017) advised adding NSEO to rainbow trout. Whereas fish that fed NSEO showed a reduction in ammonia during the storage period.
Concerning the role of TEO and NSEO as immune stimulants, the levels of lysozyme, complement 5, and IgM increased significantly (p<0.05) in all feeding groups except the groups fed 1 and 2% NSEO. Increases in immunological responses like lysozyme, complement, and immunoglobulins can thus affect fish health by increasing disease resistance.   Bipul et al. (2020), dietary supplementation with 2% NSEO significantly improved the innate immunity of O. niloticus. Enriched diets with Nigella sativa, either oil or seeds, increased immunity in rainbow trout by increasing lysozyme levels (Awad et al. 2013) and raised haemoglobin, hematocrit, and globulin levels in O. niloticus (Hussein et al. 2021).
Regarding cortisol level as a stress indicator, it significantly (p<0.05) decreased in all feeding groups except those fed NSEO, which had a lower cortisol but not significantly lower than the control. These findings were supported by (Alina et al. 2014), who discovered that 1% thyme supplementation resulted in the lowest plasma cortisol levels in O. niloticus when compared to other phytobiotics. Furthermore, Zadmajid and Mohammadi (2017) discovered that cortisol levels in blood were significantly lower in the TEO-added feed groups. Yousefi et al. (2021) demonstrated that feeding 1% NS seeds to carps resulted in a considerable drop in blood cortisol levels. The higher cortisol levels in control groups might be related to higher TAN levels as mentioned by Kuttchantran (2013), Shokr (2015) and Elsayed (2015). The current study evaluated the anti-inflammatory properties of TEO and NSEO, as well as their nano-emulsion through the expression of pro-inflammatory TNF-α and antiinflammatory TGF-β genes in the gills of feeding groups. TEO, TEO-NE, NSEO, and NSEO-NE significantly reduced pro-inflammatory TNF-α production and gene expression while they significantly increased the anti-inflammatory TGF-β expression relative to the β -actin gene. These findings were supported by Gulec et al. (2014); Valladão et al. (2019) and Mahboub et al. (2022), who discussed the dietary effect of thyme and Nigella sativa on these two genes of Nile tilapia. Gills were selected based on the findings of Lu et al. (2013), Li et al. (2015), and Liu (2021), who noticed that both of these genes were found in a variety of O. niloticus tissues but with a higher degree in gills. Up-regulated expression of TNF-α has been observed in the gills of goldfish during D. intermedius infection (Lu et al. 2013). The chemical composition of TEO and NSEO might be responsible for the mode of action. Carvacrol and thymol are the two higher chemicals in TEO (Tian et al. 2011) which have anti-inflammatory activity (Braga et al. 2006). Furthermore, thymoquinone is the main active constituent of NSEO, and its effect on decreasing pro-inflammatory TNF-α has been experimentally demonstrated to be an anti-inflammatory agent (Chehl et al. 2009). In our study, the anti-inflammatory gene was significantly down-regulated in the control group. This might be linked to the effect of higher cortisol as indicated by Castro et al. (2011). For evaluating the antibacterial properties of TEO and NSEO and their nano-emulsions, the current investigation demonstrated that TEO-NE and NSEO-NE had a superior impact on preventing streptococcosis in O. niloticus without mortality than bulk TEO and NSEO. These findings were consistent with the findings of Gultepe et al. (2014), who found that feeding Oreochromis mossambicus at 1% thyme for 45 days had a 22% cumulative mortality rate compared to 61% mortality in the control group. Furthermore, the findings were consistent with those of Salam et al. (2021), who showed that dietary TEO-NE prevented Aeromonas hydrophila infection in O. niloticus without mortality compared to 10% in groups given low concentrations of TEO. These findings might be attributable to the fact that bulk TEO and NSEO have lowered water solubility, which reduces their antibacterial action and restricts their utilization in aqueous media (Pan et al. 2014 andChang et al. 2015). Furthermore, TEO and NSEO are lipophilic biologically active compounds that are chemically and physically unstable in light and oxygen, lowering their effectiveness (Anton et al. 2008 andSanguansri &Augustin 2006,). TEO-NE and NSEO-NE might be used to solve these challenges (Rodríguez et al. 2016). Nano-encapsulation of TEO and NSEO is physically and chemically stable in aqueous media (Mason et al. 2006 andMcClements et al. 2011). According to Shaaban et al (2015) and Sharif et al. (2017), negatively charged NSEO-NE had a higher bactericidal activity than bulk NSEO due to its greater stability, regulated release, and self-assembly with cell membranes of Gram-positive bacteria followed by destruction of cellular components. Because of the evenly distributed nano-droplets, the nano-emulsion can easily enter and damage the microbial membrane (Xu et al. 2008;Di Pasqua et al. 2007). Furthermore, TEO had stronger antibacterial properties than NSEO, which could be attributed to the higher levels of thymol, p-cymene, carvacrol, eugenol, and 4-allylphenol (Lee et al. 2005;Rota et al. 2008).

Conclusions
The nano-emulsion either TEO or NSEO had the superior effect on ammonia reduction and better prevention of streptococcosis. For bulk status the TEO has the superior effect than NSEO. Also, this study could consider a promising way for application of nano-emulsion or even bulk TEO and NSEO in aquaculture.