Revolucionando a criação de tilápias com nanotecnologia para reversão sexual eficiente e sustentável

https://doi.org/10.1007/s10695-022-01156-3 Testosterone nanoemulsion produced masculinized Nile tilapia (Oreochromis niloticus) Amanda Rodrigues dos Santos Magnabosco · Ester Inácio Damião Quinova · Matheus Victor Viana de Melo · Paulo Eduardo da Silva Bastos · Thamiris Pinheiro Santos · Ivanildo Inácio da Silva Júnior · André Lucas Corrêa de Andrade · Renata Meireles Oliveira Padilha · Jadson Freitas da Silva · Fabrício Bezerra de Sá · Marília Ribeiro Sales Cadena · Pabyton Gonçalves Cadena Received: 18 March 2022 / Accepted: 27 November 2022 / Published online: 8 December 2022 © The Author(s), under exclusive licence to Springer Nature B.V. 2022 Abstract The objective of this work was to develop a food additive for the sex reversal of Nile tilapia (Oreochromis niloticus) based on a simple oil in water (O/W) nanoemulsion with testosterone propionate for incorporation into commercial feed. Oil screening and evaluation of the organoleptic and physicochemical characteristics were carried out to determine the best formulation. A palatability test was also performed. Sex reversal test was assayed using 5 experimental groups: negative control — macerated feed without hormone; free testosterone — macerated feed with 60  mg/kg of testosterone propionate diluted in ethanol; and macerated feed with testosterone propionate nanoemulsion at a concentration of 30, 60, and 90  mg/kg. Stable nanoemulsions (size 76–210  nm) with testosterone propionate were produced. All nanoemulsion-added feed was palatable to tilapia. We obtained sex reversal values of ≈65, 75, and 72% in the groups of 30, 60, and 90 mg/kg, respectively. We can conclude that the nanoemulsion showed promising results; it is capable of inducing sex reversal in tilapia, is suitable as a commercial product, and has the potential to promote safety for rural staff and reduce the environmental impact of hormones.

Keywords Sex reversal · Masculinization · Nanotechnology · Androgens Introduction Nile tilapia (Oreochromis niloticus) is the 3rd most widely produced fish in the world, just behind two carp species. In Brazil, which is the 4th largest producer in the world, 486,000 tons of tilapia were produced in 2020 (Peixe BR 2021). Tilapia meat is greatly appreciated by consumers and has a high commercial value. In addition, it is a fish that adapts easily and demonstrates great resistance to diseases (Dergal et al. 2016). Usually, male tilapia has a higher growth rate than female tilapia, and as a result, male A. R. dos Santos Magnabosco · E. I. D. Quinova · I. I. da Silva Júnior · R. M. O. Padilha · J. F. da Silva · F. B. de Sá · P. G. Cadena (*) Departamento de Morfologia E Fisiologia Animal (DMFA), Universidade Federal Rural de Pernambuco, Av. Dom Manoel de Medeiros S/N, Dois Irmãos, Recife, PE 52171-900, Brazil e-mail: pabyton.cadena@ufrpe.br M. V. V. de Melo · P. E. da Silva Bastos · M. R. S. Cadena Departamento de Biologia (DB), Universidade Federal Rural de Pernambuco, Av. Dom Manoel de Medeiros S/N, Dois Irmãos, Recife, PE 52171-900, Brazil T. P. Santos · A. L. C. de Andrade Laboratório de Imunopatologia Keizo Asami (LIKA), Universidade Federal de Pernambuco (UFPE), Av. Prof.

Moraes Rego, s/n 50780-901, Recife, Pernambuco, Brazil P. G. Cadena Laboratório de Ecofisiologia E Comportamento Animal (LECA), Universidade Federal Rural de Pernambuco, Av. Dom Manoel de Medeiros S/N, Dois Irmãos, Recife, PE 52171-900, Brazil tilapia populations increase productivity per area (Zanoni et  al. 2013). For this reason, the production of all-male populations is preferred by tilapia farmers (Abo-Al-Ela 2018). The sex reversal in tilapia is possible because this is a gonochoric species and has high sexual plasticity (Habibah et al. 2021). During the first stages of development of this species, steroidogenesis occurs, the process responsible for sexual differentiation. Due to these characteristics, it is possible to use techniques to artificially induce fish to express all the phenotypic characteristics of a male, even if their genome is programmed to differentiate into a female. After the complete absorption of the yolk sac, the animal begins to ingest food.

This is the ideal period to start introducing the feed with testosterone as sexual differentiation has not yet started (Fujimura and Okada 2007; Popma and Lovshin 1996). Among the various techniques that can be used to produce all-male populations, the exposure of larvae to androgen hormones technic has higher rates of masculinization and is easier to do (Abo-Al-Ela 2018). As a result, testosterone and its esters, such as 19-noreiltestosterone, fluoxymesterone, ethyltestosterone, 17α-methyltestosterone, dihydrotestosterone, androstenedione, trenbolone acetate, mesterolone, 17α-ethinyl testosterone, and 17α-methyl dihydrotestosterone can be used (Abo-Al-Ela 2018).

The hormonal exposure to induce sex reversal is conducted over a short period, usually up to 30 days. Also, the residual testosterone values in tilapia meat are very low, and 60  days after treatment, the values are undetectable, meaning the meat is safe for human consumption (Dergal et al. 2016). However, the handling of these hormones by tilapia farmers during the sex reversal process can contaminate them. Frequently, testosterone is acquired pure and then diluted in ethanol and manually added to the feed, which is when human contamination can occur.

This contamination can lead to several harmful health effects, such as breast and testicular cancer, poor sperm quality or function, endometriosis, and infertility (Sifakis et  al. 2017) in the affected tilapia farmers. Furthermore, the disposition and the presence of these hormones, without previous treatment, in the water bodies, cause environmental problems. Behavioral changes, infertility, and masculinization of females are some of the effects that can be induced for testosterone in nature without control (Jung et  al. 2020). To overcome these limitations to the safe use of testosterone for sex reversal, nanotechnology products such as nanoemulsions can be used.

A wide range of nanotechnology processes with applicability in aquaculture are available in the literature, and nanoemulsions are a practical and efficient alternative for this (Dar et  al. 2019). Nanoemulsions are colloidal systems resulting from the mixture of two phases, at first, immiscible, composed in most cases of an aqueous phase and an oil phase (simple water-in-oil emulsions — W/O or oil in water — O/W), stabilized with the aid of a surfactant (McClements and Jafari 2018). They are nanotechnology processes that have high stability since the nanometric size of their vesicles means they can reduce sedimentation and remain for more time in the animal’s body (McClements and Jafari 2018). The nanometric size increases the surface area, bioavailability, and solubility of lipophilic drugs, and nanoemulsions are considered one of the best carrier systems (Essa et al. 2021). The nanotechnology process is suitable for aquaculture, including the encapsulation of androgen hormones for the sex reversal of tilapia (Joshi et al. 2019). In addition, there is wide applicability of nanoemulsions in aquaculture, including therapeutic studies on the antimicrobial action of nanoemulsified substances (Swathy et  al. 2018) tested against pathogens in tilapia populations (El-Ekiaby 2019). The state of the art reveals products using nanoemulsions for aquaculture: 48 papers since 2010 and 73 patents since 2003, indicating interest in research and the development of nanoemulsions in this field (Lens 2021).

The possibility of incorporating testosterone in the oil phase of a nanoemulsion is a good alternative to the production of aquaculture products for tilapia sex reversal. The current work proposed the development of a new product that is user-friendly and more efficient in the administration of androgen hormones to tilapia and does not require direct manipulation by tilapia farmers. In this framework, the objective of this work was to develop and characterize a nanoemulsion with testosterone, incorporated in a commercial feed applied for the sex reversal of Nile tilapia.

Survival, morphometry, and gonadal histology were used to evaluate the efficiency of the nanoemulsion.

Material and methods Material Polysorbate 80 (Tween 80, EHL = 15.0), nipazole (propylparaben), and absolute ethanol were purchased from Dinâmica (Brazil), sorbitan monooleate 80 (Span 80, EHL = 4.3) was purchased from SIGMA (St. Louis, MO, USA), and commercial testosterone propionate was purchased from Ceva Hertape (MG, Brazil). Testosterone was legally purchased through registration in the Brazilian SIMPEAGRO (Integrated System of Agricultural Products and Establishments) register number R009408/2019. All other reagents used in this experiment were of analytical grade. Five vegetable oils were selected to perform a screening.

The oils used were 100% vegetables from canola, corn, cotton, soy, and sunflower, type 1 according to classification in the normative instruction no. 49/2006 of the Brazilian Ministry of Agriculture, Livestock and Food Supply (MAPA 2006).

Preparation of nanoemulsions The type of vegetable oil, proportion of surfactants, and the presence or absence of propylparaben were evaluated in this first screening. The prepared nanoemulsions were identified from two letters of each oil name: canola — CA, corn — CO, cotton — CT, soy — SO, and sunflower — SF, followed by numbers corresponding to the variation in the surfactants and the presence or absence of propylparaben, being 1 — 5% Span, 15% Tween, propylparaben present; 2–10% Span, 10% Tween, propylparaben present; 3–15% Span, 5% Tween, propylparaben present; 4–5% Span, 15% Tween, propylparaben absent; 5–10% Span, 10% Tween, propylparaben absent; and 6–15% Span, 5% Tween, propylparaben absent.

The oil/water (O/W) nanoemulsions were prepared using a high-energy emulsification method by magnetic stirring from an oil phase (O) produced from a mixture of 10% (w/w) vegetable oil (cotton, canola, sunflower, corn, or soy), 5–15% (w/w) Span 80, and 2–6% (w/w) testosterone propionate (TP) solution. The TP concentration is based on the dosage recommended in the feed (60  mg/kg) for tilapia sex reversal (Zanardi et  al. 2011). The O phase was first homogenized using a magnetic stirrer for 5 min at 7 g at room temperature (25 ± 2  °C). Simultaneously in another container, the water phase (W) was prepared with 64% distilled water (w/w) and 5–15% (w/w) of Tween 80. The W phase was first homogenized for 5  min at 7  g and then mixed with 40  mg of propylparaben dissolved in 1  mL of absolute ethanol. This W phase remained for a further 5 min at 7 g for total evaporation of the ethanol. Propylparaben can act as a preservative in the formulation. Finally, the O phase was dripped (flow of 4  mL/min) using a burette into the W phase under magnetic stirring at 7  g to form the O/W nanoemulsion. The dispersion was strongly stirred at 60 g for 24 h to obtain the nanometric size.

Characterization of nanoemulsions Macroscopic physical analysis The formulations were evaluated for their organoleptic characteristics immediately after preparation for appearance — phase of dispersion: homogeneous (HM) or heterogeneous (HT); appearance — consistency: fluid (FL), slightly thick (ST), thick (TH), or very thick (VT); appearance — opacity: translucent (TR), little opaque (LO), or opaque (OP); appearance — lumps: present (PR) or absent (AB); odor: present (PR) or absent (AB); and color: present (PR) or absent (AB) (ANVISA 2004; Damasceno et al. 2016;

Frange and Garcia 2009).

Stability tests The nanoemulsions were stored in sterile sealed containers with a headspace equivalent to 1/3 of the total volume of the container to allow the gas exchange of the nanoemulsion. The samples were submitted to centrifugation after preparation at 1210  g for 30  min (Kacil CE-01 Centrifuge) followed by the analysis of the appearance — dispersion phase evaluation:

homogeneous (HM) or heterogeneous (HT). Samples of nanoemulsions were submitted to thermal stress tests for 24 h to evaluate cooling (5 ± 2 °C), freezing (− 20 ± 2  °C), and heating (37, 40, and 50 ± 2  °C).

After the thermal stress tests, the macroscopic physical analysis (item 2.3.1) of each nanoemulsion was evaluated again at room temperature (25 ± 2  °C).

These results were compared with the previous results of macroscopic physical analysis (item 2.3.1).

The stable nanoemulsions were submitted to physicochemical characterization.

Physicochemical characterization The nanoemulsions were diluted (50 ×) in doubledistilled water before analysis to reduce the turbidity.

The average size (nm), polydispersity index (PDI), and zeta potential (mV) of the oil vesicles were evaluated using the standard photon correlation spectroscopy (PCS) technique fixed at 90 to 25  °C using a Zetasizer Nano ZS (Malvern, UK) (Cadena et  al.

2013; Pereira et al. 2018; Riquelme et al. 2019). Samples of empty (E) nanoemulsions (without hormone) and with TP in weight of 20 to 60  mg in 10  g of nanoemulsion were tested. The data were measured in triplicate.

Incorporation of nanoemulsions in a commercial feed The palatability test was assayed using a mixture in the ratio of 2:1:1 respectively as feed (commercial extruded macerated feed — 40% crude protein):distilled water:empty nanoemulsion of each oil (canola, corn, and soy). This modified feed was repelled again and stored for 24  h at room temperature (25 ± 2  °C) for drying and then offered to juvenile animals.

The TP nanoemulsion-added feed was prepared through the incorporation of TP nanoemulsions in the commercial extruded macerated feed (40% crude protein) using a ratio of 100:10:0.5 — 1.5 (0.5 = 30 mg/ kg, 1.0 = 60 mg/kg, and 1.5 = 90 mg/kg), respectively of macerated feed:water:TP nanoemulsion. The mixtures were homogenized and stored for 4  h at room temperature (25 ± 2  °C) for drying and then offered to larvae animals. Finally, the feed of conventional treatment (free testosterone — FT group) was produced by dissolving 0.6 g of TP in 10 g absolute ethanol and adding 100  g of macerated feed to obtain a 60 mg/kg concentration. This feed was stored for 4 h at room temperature (25 ± 2 °C) for complete ethanol volatilization.

Experimental fish The experiments were carried out at the Laboratório de Ecofisiologia e Comportamento Animal — LECA, Universidade Federal Rural de Pernambuco — UFRPE. All protocols presented here were submitted to and approved by the Ethics Committee on the Use of Animals of UFRPE, protocol number 117/2019.

The juveniles and larvae of Nile tilapia were obtained from a local fish farm (Estação de Aquicultura Continental Professor Johei Koike) at the same University.

All animals used in this experiment were maintained under the same conditions: 14/10 h photoperiod (light/dark), the temperature of 27 ± 1  °C, and pH of 7.0 ± 0.5. The juveniles were housed in aquariums with a capacity of 80 L (item 2.5.1), and larvae were housed in aquariums with a capacity of 10 L (item 2.5.2). The water of the sex reversal test was partially renewed three times per week by siphoning off at least 40% of the volume and adding new water.

Residues generated in the experiments were treated by the advanced oxidative process in a reactor, using photooxidative UV and H O (Hansen and Andersen 2012), and then discarded.

Palatability test with juveniles Three different feeds containing canola, corn, and soy empty nanoemulsions (item 2.4) were offered ad libitum to 30 juvenile Nile tilapia with an average weight of 10.5  g in a proportion of 0.5  g of feed per individual. Feeds were provided for two consecutive days and at different times a day (morning — canola, afternoon — corn, and night — soy) to the fish to prove its palatability. The animals were not fed other diets during the test.

Sex reversal using TP nanoemulsions In total, 550 larvae with an average size of 10  mm were used, approximately 12-day postfertilization (dpf). They were previously acclimatized until complete absorption of the yolk sac. Subsequently, they were separated into 5 groups of 55 animals in duplicate (total 110 animals per experimental group) kept under the same conditions in aquariums with a capacity of 10 L. The animals were fed 6 times a day (8 am, 10 am, 12  pm, 2  pm, 4  pm, and 6  pm) with the following treatments: (i) negative control — macerated feed without hormone (NC group); (ii) free testosterone — macerated feed with 60  mg/kg of TP diluted in ethanol (conventional treatment) (FT group); (iii) macerated feed with TP nanoemulsion at a concentration of 30  mg/kg (30  T group); (iv) macerated feed with TP nanoemulsion at a concentration of 60  mg/ kg (60  T group); and (v) macerated feed with TP nanoemulsion at a concentration of 90  mg/kg (90  T group). The hormonal induction had a duration of 30  days (12–42 dpf) and was divided into two periods of 15  days. In the first 15  days, the animals in each aquarium were fed 0.160  g of respectively feed 6 times a day, and 2/5 of the total volume of water in the aquarium was renewed 3 times a week. Due to the growth of the animals, during the second 15 days, the animals in each aquarium were fed 0.320 g of respectively feed 6 times a day, and 3/5 of the total volume of water in the aquarium was renewed 3 times a week.

The animals were fed for a further 5  days with only macerated feed without hormone and then euthanized 35 days after the beginning of the hormonal induction (47 dpf) through immersion in water with an anesthetic alcoholic solution (Eugenol 300  mg/L diluted in 1 mL of absolute ethanol) (Vidal et al. 2008). The scheme of experimental design is shown in Fig. 1.

Morphometry and survival rate of animals The morphometry (110 per experimental group) was carried out after euthanasia using a caliper rule in the lateral view of the fish. The following parameters were collected: total length (head–tail direction), head height (dorsoventral direction), and eye diameter. The survival rate was determined by the difference between the initial and final numbers of animals in each aquarium. The parameters measured are shown in Fig. 2.

Collection of gonads and processing of histological material Immediately after measuring the morphological parameters, an opening was made in the coelomic cavity, and the fish were immediately fixed in Davidson solution (70% ethanol, acetic acid, and paraformaldehyde) for 48 h to preserve the gonads. Subsequently, the animals were stored in 70% ethanol at room temperature (25 ± 2  °C). Later, the head, tail, dorsal fin, spine, and viscera were removed. Part of the coelomic cavity was preserved to not cause injury in gonads. The animals were dehydrated by increasing ethanol concentrations (70 to 100%) and butanol for 15  min and then processed histologically for inclusion in paraffin (n = 20 per experimental group). Blocks were cut transversely using a manual microtome (5  μm) (Leica RM2125 RTS). Transversal sections were maintained in a water bath and placed on slides (37  °C). Finally, deparaffinization of the slides was performed by heating to 55 °C for 12 h and proceeded to the slide staining stage using the hematoxylin and eosin protocol. At the end of the process, the slides were mounted with Etellan for later analysis.

Fig. 1 The scheme of experimental design to induce sex reversal related to fish (Oreochromis niloticus) development stages Fig. 2 Parameters to measure morphometry with the aid of a caliper in the lateral view of the fish. TL, total length (head– tail direction); HH, head height (dorsoventral direction); ED, eye diameter

Sexing by histological evaluation of the slides A structural evaluation of gonad tissue was performed to determine sex and study gonadal development using an optical microscope with LED light (BIO2B SSI).

For qualitative classification, the animals were considered male when gonad tissue contained only male germinative cells (MGC), female when gonad tissue contained only female germinative cells (FGC), or ovotestis when gonad tissue presents male and female germinative cells at the same time. We consider qualitatively predominantly male ovotestis or predominantly female ovotestis when we observed a ratio of germinative cells of at least 5:2 (MGC:FGC or vice versa).

When the ratio between MGC and FGC was less than 5:2 within the same gonad, it was classified as undefined ovotestis.

Statistical analysis The data of macroscopic characterization of nanoemulsions were qualitatively analyzed according to ANVISA guidelines (ANVISA 2004) that were based on ICH stability testing of new drug substances (2003).

Statistical planning for the assays with fish was conducted in authentic duplicate using 55 animals per aquarium per experimental group. Each animal was considered a replicate.

All data of physicochemical characterization of nanoemulsions and morphometric parameters for the animals were analyzed by one-way ANOVA (Origin Lab. Northampton, MA, USA). When they were significant, the Tukey test with p < 0.05 was applied.

Results Macroscopic physical analysis The organoleptic characteristics of the nanoemulsions were evaluated considering their visual appearance and are shown in Table 1. All formulations were considered homogeneous, which is an indication of stability. It was also observed that the formulations with 5% (w/w) Span 80 in their composition, with and without propylparaben (CT1, CT4, CA1, CA4, SF1, SF4, CO1, CO4, SO1, and SO4), were considered more fluid compared to the other formulations.

In addition, when the span 80 concentration was increased (10% and 15%), they became slightly thick (CT3 and CT6), thick (CA6, SF6, CO6, and SO6), or very thick (CA3, SF3, CO3, and SO3). Regarding opacity, it was noted that nanoemulsions containing propylparaben were considered opaque when compared to nanoemulsions without preservatives, except the SO1 formulation, which was considered low opaque. Only the SO5 formulation had lumps.

No nanoemulsions presented odor or color. Formulations without propylparaben proved to be more translucent and less thick compared to formulations that contained the preservative. However, formulations without propylparaben were discarded in future experiments because the preservative is more suitable to prevent biological contamination.

Preliminary stability assessment Immediately after their preparation, the nanoemulsions were submitted to stability evaluation by centrifugation, and no changes were observed in their organoleptic characteristics or phase separation compared to the newly prepared formulation.

The results are shown in Table 2. The nanoemulsions submitted to cooling (5 ± 2 °C) for 24 h did not show any changes when returning to room temperature (25 ± 2  °C). However, when submitted to freezing (− 20 ± 2 °C) for 24 h, the SO1 formulation was opaque after defrosting compared to the other formulations. The nanoemulsions submitted to heating at 37 and 40 ± 2  °C showed no changes, except for the SO1 formulation, which after being heated at temperatures of 37, 40, and 50  °C was considered opaque when compared to before heating (slightly opaque). However, at 50 ± 2  °C, all formulations with 15% w/w of Tween 80 (CT1, CA1, SF1, CO1, SO1) were considered the most fluid (Table 1) presenting phase separation (Table 2) and were discarded from further tests. CT2 and SF2 formulations (10% w/w of Tween 80) also had phase separation at 50  °C (Table 2) and were also discarded from further tests. The nanoemulsion considered more fluid was easier to produce under magnetic stirring.

Because the nanoemulsions with 15% w/w Span 80 were considered thicker (Table 1), formulations CA2, CO2, and SO2 with 10% w/w Span 80 were preferred to the others and were chosen for the subsequent experiments.

Physicochemical characterization The results of the physicochemical characterization of the nanoemulsions are shown in Table 3. Based on the results, nanoemulsions had an average size between 76.02 and 210.10 nm. The presence of TP in the formulation increased the vesicle size in a directly proportional manner (p < 0.05). Regarding the PDI, values between 0.30 and 0.56 were obtained, indicating that the high testosterone concentration used increased the PDI and decreased the homogeneity of the formulations. Regarding the zeta potential, it was found that the nanoemulsions had a negative charge, with values between − 49.10 and − 38.60.

Table 1 Evaluation of the organoleptic characteristics of oil/ water (O/W) nanoemulsions containing testosterone propionate (TP) regarding the type of vegetable oil used, the proportions of Span 80 and Tween 80, and the presence or absence of propylparaben immediately after preparation (ANVISA 2004;

Damasceno et al. 2016; Frange and Garcia 2009) Subtitles: Appearance — phase of dispersion: homogeneous (HM) or heterogeneous (HT); appearance — consistency: fluid (FL), slightly thick (ST), thick (TH), or very thick (VT); appearance — opacity: translucent (TR), little opaque (LO), or opaque (OP);

appearance — lumps: present (PR) or absent (AB); odor: present (PR) or absent (AB); color: present (PR) or absent (AB) OilSampleSpan 80 (w/w)Tween 80 (w/w)PropylparabenAppearanceOdorColor Phase of dispersion ConsistencyOpacityLumps CanolaCA15%15%PRHMFLOPABABAB

CA210%10%PRHMSTOPABABAB

CA315%5%PRHMVTOPABABAB

CA45%15%ABHMFLTRABABAB

CA510%10%ABHMFLLOABABAB

CA615%5%ABHMTHOPABABAB

CornCO15%15%PRHMFLOPABABAB

CO210%10%PRHMSTOPABABAB

CO315%5%PRHMVTOPABABAB

CO45%15%ABHMFLTRABABAB

CO510%10%ABHMFLLOABABAB

CO615%5%ABHMTHOPABABAB

CottonCT15%15%PRHMFLOPABABAB

CT210%10%PRHMSTOPABABAB

CT315%5%PRHMSTOPABABAB

CT45%15%ABHMFLTRABABAB

CT510%10%ABHMSTOPABABAB

CT615%5%ABHMSTOPABABAB

SoySO15%15%PRHMFLLOABABAB

SO210%10%PRHMSTOPABABAB

SO315%5%PRHMVTOPABABAB

SO45%15%ABHMFLTRABABAB

SO510%10%ABHMSTOPPRABAB

SO615%5%ABHMTHOPABABAB

Sunflower

SF15%15%PRHMFLOPABABAB

SF210%10%PRHMPEOPABABAB

SF315%5%PRHMVTOPABABAB

SF45%15%ABHMFLTRABABAB

SF510%10%ABHMFLLOABABAB

SF615%5%ABHMTHOPABABAB

Canola and corn formulations demonstrated the best results in comparison with soy formulations in all concentrations of TP (smaller size, smaller PDI, and higher zeta potential); however, the soy nanoemulsion with 60  mg TP was chosen due to the cost–benefit characteristics of this oil, as it is easy to acquire in a large quantity at a low cost.

In addition, the nanoemulsion with 60  mg TP was better than the others for producing the feed; it is possible to reduce the production costs because a smaller amount of nanoemulsion (10 g) was used to produce 100 g of feed with 60 mg/kg (60 T) of TP proportion. In addition, the values of macroscopic characteristics and zeta potential of 60 mg of TP in Table 2 Evaluation of the organoleptic characteristics of oil/water (O/W) nanoemulsions containing testosterone propionate (TP) after heat stress (50 ± 2 °C) returning to room temperature (ANVISA 2004; Damasceno et al. 2016; Frange and Garcia 2009) Subtitles: Appearance — phase of dispersion:

homogeneous (HM) or heterogeneous (HT);

appearance —consistency:

fluid (FL), slightly thick (ST), thick (TH), or very thick (VT); appearance — opacity: translucent (TR), little opaque (LO), or opaque (OP); appearance — lumps: present (PR) or absent (AB); odor: present (PR) or absent (AB); color:

present (PR) or absent (AB) Heating 50 ± 2 °C OilSample Appearance OdorColor Phase of dispersion ConsistencyOpacityLumps CanolaCA1HTFLOPABABAB

CA2HMSTOPABABAB

CA3HMVTOPABABAB

CornCO1HTFLOPABABAB

CO2HMSTOPABABAB

CO3HMVTOPABABAB

CottonCT1HTFLOPABABAB

CT2HTSTOPABABAB

CT3HMSTOPABABAB

SoySO1HTFLOPABABAB

SO2HMSTOPABABAB

SO3HMVTOPABABAB

Sunflower

SF1HTFLOPABABAB

SF2HTSTOPABABAB

SF3HMVTOPABABAB

Table 3 Effect of testosterone propionate (TP) in oil-in-water (O/A) nanoemulsions of canola, corn, and soy oil on the physicochemical parameters as average size (mm), polydispersity index (PDI), and zeta potential ζ (mv) Experimental groups were compared by one-way ANOVA (average size for canola (F(3.11) = 223.92, p < 0.001), corn (F(3.11) = 542.13, p < 0.001), and soy F(3.11) = 732.70, p < 0.001 and ζ (mv) for canola F(3.11) = 13.99, p < 0.001), corn (F(3.11) = 67.12, p < 0.001), and soy F(3.11) = 180.70, p < 0.001)) followed by Tukey test with *p < 0.05 OilProduced formulationAverage size (nm)PDIζ (mv) CanolaEmpty76.02 ± 3.050.372 − 42.10 ± 1.64 20 mg TP141.00 ± 5.73*0.304 − 43.90 ± 0.65 40 mg TP157.70 ± 4.80*0.360 − 45.60 ± 0.55* 60 mg TP148.40 ± 2.97*0.462 − 47.10 ± 0.76* CornEmpty87.52 ± 0.980.301 − 40.60 ± 0.11 20 mg TP125.50 ± 0.70*0.331 − 38.60 ± 1.08 40 mg TP154.50 ± 1.46*0.316 − 42.70 ± 0.82 60 mg TP173.50 ± 5.24*0.541 − 48.50 ± 1.19* Soy Empty86.11 ± 1.610.320 − 32.60 ± 0.95 20 mg TP138.40 ± 2.13*0.304 − 41.70 ± 1.36* 40 mg TP146.00 ± 0.98*0.298 − 41.50 ± 0.52* 60 mg TP210.10 ± 5.85*0.559 − 49.10 ± 0.06* the soy oil formulation (≈ − 49.1  mV) could indicate good stability. Finally, as our choice was based on industrial production, and not simply on the best physicochemical characteristics, we tested soy nanoemulsions in further experiments.

Palatability test with juveniles The feed containing canola, corn, and soy empty nanoemulsions was offered ad  libitum to juveniles with 100% palatability of all feeds on two consecutive days. As cited above, we considered the use of soy nanoemulsion for sex reversal.

Morphometry and survival rate of animals Larvae were used for sex reversal; 35 days after the beginning of the hormonal induction (47 dpf), the animals were euthanized. The values of total length, head height, and eye diameter of the animals are shown in Table 4. We observed a reduction in the values of total length and head height in the 60  T and 90  T groups (≈10%) compared to the other experimental groups (Table 4). However, as the animals did not present modifications in eye diameter values, we considered that the observed morphometric alterations are probably transitory. The survival rate values are expressed in Table 5. In all groups tested, the survival rate remained above 70%. The 30  T group had the lowest mortality rate (5.45%).

Based on these results, the use of nanoemulsions is considered safe for the masculinization of tilapia.

Quantification of sexually reversed animals by histological evaluation of the gonads Typical gonads identified in our work are shown in Fig. 3, and it was possible to observe male and female germinative cells in different development stages. Using this method, it was possible to identify and classify qualitatively each gonad after the finalization of the sex reversal experiments. Results obtained for each experimental group are shown in Table 6. In general, we had a high incidence of ovotestis and did not observe female gonads. We observed sex reversal in all experimental groups and a high number of male gonads in the free testosterone, 60  T and 90  T groups. However, a decrease in the number of these male gonads was observed when the sex reversal was realized using nanoemulsions. Considering that predominantly male ovotestis and some undefined ovotestis produce male fish at the end of their maturation, there was an increase in the sex reversal of ≈90% for the free testosterone group and ≈65, 75, and 72% in the groups of 30 T, 60 T, and 90 T, respectively.

Discussion We developed a food additive based on a simple oil in water (O/W) testosterone nanoemulsion for Table 4 Evaluation of the total length (head–tail direction), head height (dorsoventral direction), and eye diameter of the tested animals in lateral view after sex reversal in Nile tilapia Groups: negative control (NC — feed without hormone); free testosterone diluted in ethanol added to feed (FT — 60 mg/kg);

testosterone propionate (TP) nanoemulsion added to feed with 30 mg/kg (30 T), 60 mg/kg (60 T), and 90 mg/kg (90 T) concentrations Experimental groups were compared by one-way ANOVA (total length F(4, 475) = 8.74, p < 0.001, head height F(4,

476) = 5.86, p < 0.001, eye diameter F(4, 476) = 1.02,

p = 0.396) followed by Tukey test with *p < 0.05 GroupAverage (cm) Total lengthHead heightEye diameter NC3.51 ± 0.660.90 ± 0.220.32 ± 0.32 FT3.41 ± 0.600.89 ± 0.190.29 ± 0.49 30 T3.41 ± 0.460.85 ± 0.120.30 ± 0.04 60 T3.14 ± 0.68*0.81 ± 0.17*0.28 ± 0.06 90 T3.09 ± 0.60*0.80 ± 0.17*0.28 ± 0.05 Table 5 Evaluation of the survival rate in Nile tilapia of the tested groups Groups: negative control (NC — feed without hormone); free testosterone diluted in ethanol added to feed (FT — 60 mg/kg);

testosterone propionate (TP) nanoemulsion added to feed with 30 mg/kg (30 T), 60 mg/kg (60 T), and 90 mg/kg (90 T) concentrations GroupsSurvival rate (%) NC82.73 FT84.55 30 T94.55 60 T85.45 90 T87.27 incorporation into commercial fish feed, with efficiency verified for the sex reversal of tilapia. There are few studies using nanotechnology processes to obtain male tilapia. In this context, Joshi et  al.

(2019) used an aromatase inhibitor encapsulated in poly lactic-co-glycolic acid (PLGA) nanoparticles, obtaining sex reversal higher than 89%. The percentage of reversed animals in our study was above 55%;

however, using the best nanoemulsion, we obtained ≈75% of reversed animals. Our histological findings confirmed that predominantly female ovotesits and predominantly male ovotestis can be developed into functional males, using our treatments to induce sex reversal. Comparison of the micromorphology of the treated (FT, 30 T, 60 T, and 90 T groups) vs. negative control revealed different developmental stages of sex differentiation at the same time (47 dpf). However, the FT and 60 T groups had the best values for functional males (Table 6).

The type and concentration of androgens interfere with the efficiency of sex reversal. Celik et  al.

(2011) studied the variation in concentrations of 17α-methyltestosterone and obtained sex reversal of 69.4% and 93.7% with 30  T and 60  T groups, respectively. A similar value was obtained in our Fig. 3 Typical histological observations of gonads after sex reversal stained with hematoxylin and eosin. Groups: negative control (NC, feed without hormone), free testosterone diluted in ethanol added to feed (FT, 60 mg/kg), and testosterone propionate (TP) nanoemulsion added to feed with 30 mg/ kg (30 T), 60 mg/kg (60 T), and 90 mg/kg (90 T) concentrations. Subtitle:

Undefined ovotestis gonads (A), predominantly male ovotestis (B), male gonads (C), and predominantly female ovotestis (D).

Female germinative cells are indicated by arrows and male germinative cells are indicated by stars. Magnification: × 1000 Table 6 Percentage of reversed animals (males), females, and ovotestis (undefined, predominantly males, and predominantly females) in Nile tilapia Groups: negative control (NC — feed without hormone); free testosterone diluted in ethanol added to feed (FT — 60 mg/kg);

testosterone propionate (TP) nanoemulsion added to feed with 30 mg/kg (30 T), 60 mg/kg (60 T), and 90 mg/kg (90 T) concentrations GroupsMaleFemaleUndefined Ovotestis Predom.

male ovotestis Predom.

female ovotestis NC--30%10%60% FT60%--30%10% 30 T35%-40%10%15% 60 T50%-10%20%20% 90 T45%-35%10%10%

study for 30  T (65% sex reversal). Androgens in general can be used for tilapia sex reversal (Abo- Al-Ela 2018); however, 17α-methyltestosterone is still more effective than the other options (Golan and Levavi-Silvan 2014). Although TP was effective for sex reversal in our study, the percentage of male fish was lower than those normally obtained with 17α-methyltestosterone. For this reason, future studies to improve the percentage of reversed animals including 17α-methyltestosterone should be conducted.

Another important factor in the efficiency of sex reversal is to administer testosterone to the animal at the ideal period. The ideal period for testosterone administration is before the animal begins sexual differentiation when the animal absorbs the yolk sac and begins to eat. This occurs approximately between 12–13 dpf (Fujimura and Okada 2007; Popma and Lovshin 1996). From 17 to 32 dpf, the phenotypic sex is fixed. From 30 dpf, the sexual differentiation begins, and it is possible to observe the first morphological changes in the gonads. From 42 dpf, the morphological changes become even more evident, due to the increased expression of 2 genes, Dmrt1 gene (testis) and Cyp19a1a gene (ovary), responsible for sex differentiation (Tao et  al. 2018). In our experiment, testosterone was administered in the ideal period for sex reversal, and for this reason, we obtained ≈65, 75, and 72% in the groups of 30  T, 60  T, and 90  T values for sex reversal, respectively. In addition, we obtained 100% palatability when providing food to the animals. Neither mortality nor behavioral changes were observed qualitatively, suggesting the safety of administering the new product. Our results are promising, but not conclusive because we obtained a high percentage of ovotestis. The quantity of female or male germinative cells added to the ideal period of administration of testosterone is essential to the success of sex reversal. In our results, the classification “predominantly female ovotestis” or “predominantly male ovotestis” is not a conclusion, but that this animal has a high possibility of differentiating into female or male fish. Based on this, future studies can be conducted for a longer time to confirm the total gonad differentiation.

The results of our preliminary stability and temperature tests are consistent with the standards proposed by the ANVISA (2004) guidelines. In addition, the evaluation of these characteristics showed the possible stability of our nanoemulsions. We obtained a mean size of 76–210 nm, and these sizes were classified as nanoemulsions since the values were less than 300  nm (Anton and Vandamme 2011; McClements and Jafari 2018). We observed differences in the vesicle size compared to the control group; the high testosterone concentration increased the vesicle size. The polydispersity index (PDI) and zeta potential are important factors to evaluate the stability of nanoemulsions. PDI values determine the homogeneity of the formulation by evaluating the vesicle size.

According to Danaei et  al. (2018), values below 0.2 are preferable as they indicate low vesicle size variation. However, there is no safe or acceptable value limit; PDI values can be higher or lower depending on the purpose of the formulation. The results obtained in our study correspond to what was expected; all values remained below 0.6. Values of zeta potential indicate surface charge and electrostatic parameters of vesicles (Grisham and Nanda 2020). The produced nanoemulsions showed high negative values of zeta potential, indicating a high magnitude of the interaction of the repulsion forces between the vesicles and demonstrating their stability (Caddeo et al. 2008).

All reversed fish presented growth values considered normal and corresponding to age, that is, at least 3  cm (Kubtza 2006). We observed significant differences in total length and head height in the 60 T and 90  T groups compared with other groups. However, the animals remained healthy and continued growing as expected. No significant differences were found in the eye diameter. During the period following absorption of the yolk sac, there is massive cannibalism in tilapia populations. These values can vary between 10 and 30% of the fry population (Macintosh and Little 1995). In our experiments, we observed mortality of less than 30%, similar values to those found in tilapia populations.

Tilapia sex reversal has an economic interest because tilapia is one of the most consumed fish species nowadays, and these values are expected to increase over the years. Nile tilapia is the 3rd most produced fish species in the world. Brazil is the 4th largest producer in the world, and the volume of production increases every year. It has been observed that consumption per person has also been growing every year (Peixe BR 2021). This is because tilapia meat is a protein of high biological value and is well appreciated by the population. In addition, it is quick to produce and cheaper compared to other proteins, proving to be a great alternative for developing and underdeveloped countries that suffer from hunger. The use of the developed product can have great advantages over the traditional route, such as safety for tilapia farmers, and the environment.

Based on our results, other advantages are that the product is user-friendly for administration to the animals, time optimization for the sex reversal procedure, and the guarantee of the correct dosage of the used hormone. The reagents are safe, and equipment costs are low, and the production method is simple, which makes large-scale production economically viable and means it is possible to apply prices that are competitive with other available feeds. Soybean oil, the lipid chosen for testosterone incorporation, is one of the cheapest and most easily accessible vegetable oils available nowadays.

Conclusion In summary, it is possible to conclude that the nanoemulsion showed promising results, demonstrating its ability to perform sex reversal in Nile tilapia fingerlings in the tested concentrations. Future studies to improve the percentage of reversed animals for a longer time should be conducted. The concentration that presented the most satisfactory results was the 60  T group (60  mg/kg of TP). Its use is promising since it can be included in the composition of feed for testosterone release. This proved to be a safer and more efficient method for the administration of this hormone for tilapia sex reversal in aquaculture.

The increased safety is mainly due to the provision of a finished product for purchase, which does not require direct manipulation of testosterone. Finally, the product is produced using a cheap technique that can easily be produced on a large scale.

Acknowledgements The authors would like to thank the Universidade Federal de Pernambuco for the availability of Laboratórios Associados em Rede de Nanotecnologia (LAR- Nano), and all the members involved in this research.

Author contribution Details of each author with their contribution in this paper: Amanda Rodrigues dos Santos Magnabosco — conceptualization, methodology, validation, formal analysis, investigation, data curation, writing — original draft, writing — review and editing, and visualization; Ester Inácio Damião Quinova — investigation and data curation; Matheus Victor Viana de Melo — investigation and data curation; Paulo Eduardo da Silva Bastos — investigation; Thamiris Pinheiro Santos — investigation; Ivanildo Inácio da Silva Júnior — investigation; André Lucas Corrêa de Andrade — formal analysis and data curation; Renata Meireles Oliveira Padilha — investigation and writing — original draft; Jadson Freitas da Silva — investigation; Fabrício Bezerra de Sá — investigation; Marília Ribeiro Sales Cadena — supervision, project administration, writing — original draft, and writing — review and editing; and Pabyton Gonçalves Cadena — conceptualization, methodology, validation, formal analysis, resources, data curation, writing — original draft, writing — review and editing, visualization, supervision, project administration, and funding acquisition. All authors read and approved the final manuscript.

Funding The authors would like to thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior — CAPES, for the scholarship to Amanda R. Santos-Magnabosco, Ester I. D. Quinova, and Jadson F. Silva; CNPq (National Council for Scientific and Technological Development) for the PIBITI scholarship for Matheus V. V. Melo and DT-2 productivity scholarship (Process 310401/2017–8) for Pabyton G.

Cadena; and Universidade Federal Rural de Pernambuco (Call 015/2018) and Serviço Brasileiro de Apoio às Micro e Pequenas Empresas (SEBRAE) (Catalisa ICT No. 29083*198) for the financial support.

Data Availability The data and material used in this study are available from the corresponding author on request.

Code availability Not applicable.

Declarations Ethics approval All protocols presented in this study were submitted and approved by the Ethics Committee on the Use of Animals of Universidade Federal Rural de Pernambuco — UFRPE, protocol number 117/2019.

Consent to participate Not applicable.

Consent for publication Not applicable.

Conflict of interest The research reported in this manuscript is part of a patent (BR102020004629-2) in the course of the process.

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RESEARCH

Nanoemulsion containing 17α‑methyltestosterone as a feed additive showed high sex reversal efficiency in Nile tilapia (Oreochromis niloticus) Matheus Victor Viana de Melo · Amanda Rodrigues dos Santos‑Magnabosco Jadson Freitas da Silva · Aline Rafaely Damaso Alves · Hansheys Ménard Stefanny Arielle Gomes da Silva · Dijaci Araújo Ferreira Pabyton Gonçalves Cadena Received: 10 July 2025 / Accepted: 28 October 2025 © The Author(s), under exclusive licence to Springer Nature Switzerland AG 2025 Abstract This study was aimed to produce and characterize a nanoemulsion containing encapsulated 17α-methyltestosterone (17-αMT) as a feed additive to tilapia for sex reversal. A complete experimental design was carried out to evaluate the influence of soybean oil, Span 80, and Tween 80 surfactants on the organoleptic and physicochemical parameters of nanoemulsions. The best formulation was incorporated into the feed and offered to the tilapia fingerlings. The animals were monitored in three different periods (sex reversal, rearing, and fattening) and divided into four groups, i.e., CON (control, diet without feed additive), 17-αMT + L (diet with commercial additive), NE30, and NE60 (diet with nanoemulsion containing 30 and 60  mg of 17-αMT/kg, respectively), and at the end their gonads were analyzed to confirm sex. Based on the Pareto analysis, higher amounts of Span 80 and Tween 80 decreased vesicle size and increased zeta potential. Based on this, the F5 and F9 formulations had the best stability and physicochemical parameters, with F9 being selected as the best with vesicle size of 102.21 ± 30.35  nm, PDI with 0.287, and zeta potential of –38.2 ± 1.56  mV. CON, 17-αMT + L, NE30, and NE60 groups obtained, respectively, 65.52%, 99.17%, 99.26%, and 93.98% of masculinization. The zootechnical parameters and survival after three different periods were monitored and not affected by nanoemulsions, indicating that they have low toxicity. Therefore, it was possible to produce and characterize nanoemulsions containing encapsulated testosterone showing high sex reversal efficiency (> 99%) reducing by 50% the concentration of testosterone commonly used in this technique.

Handling Editor: Pierre Boudry

* Pabyton Gonçalves Cadena

pabyton.cadena@ufrpe.br Departamento de Morfologia E Fisiologia Animal (DMFA), Universidade Federal Rural de Pernambuco. Av., Dom Manoel de Medeiros S/N, Dois Irmãos, Recife, PE 52171-900, Brazil

Departamento de Pesca E Aquicultura, Universidade Federal Rural de Pernambuco. Av., Dom Manoel de Medeiros S/N, Dois Irmãos, Recife, PE 52171-900, Brazil 656 Page 2 of 19 Keywords Nanotechnology · Male hormone · Masculinization · Aquaculture Introduction Animal production systems are relevant in economic development and in ensuring global food security, attracting more investments in technology and innovation. As an example, the global fisheries and aquaculture industry reached, in 2022, the production of 185.4 million tons of aquatic animals (FAO 2024). In this sector, aquaculture accounted for 94 million tons, representing 51% of total production, surpassing capture fisheries for the first time, according to FAO report (2024). Among animals produced, fish represented the most prominent group, with the cultivation of tilapia (Oreochromis niloticus) being highly relevant (Asad et  al. 2023; FAO 2024). Its resistance to disease, rapid growth, omnivorous feeding habits, feed conversion efficiency, and adaptability to diverse breeding systems and aquatic environments has made the use of this fish an attractive choice for producers (PEI- XEBR 2025; Ramirez et al., 2024).

In tilapia farms, there is a preference for male animals to the detriment of females, due to better zootechnical parameters and uniformity of batches, ensuring better feed conversion in the fattening phase and consequently more economic gain (Ramirez et  al. 2024).

In this way, producers choose to create a male monosex batch, using the application of various sex reversal technologies (Melo et  al. 2022). The main one is the use of hormonal additives in commercial feed, where the most usual form is the addition of 60  mg of 17α-methyltestosterone (17-αMT) per kilogram of feed (Abo-Al-Ela 2018; Sarker et  al 2022). However, 17-αMT is a hydrophobic compound, which limits its bioavailability by fish (Yostawonkul et  al 2023). To make the application viable, fish farmers dilute the 17-αMT in oil or alcohol, which can affect the palatability and texture of the feed. In addition, 17-αMT presents a risk of bioaccumulation in the environment and potential toxic effects on human and other animal health (Abo-Al-Ela 2018; Yostawonkul 2023).

In the literature, there are few studies that specifically name the commercial hormonal additives used in tilapia farming. In Brazil, the 4th largest producer of tilapia in the world (FAO 2024), there is only one product licensed for sale in the country, according to the Panorama da Aquacultura Magazine (2018). This product is composed only of 17-αMT with a lactose vehicle, requiring oil for dilution and incorporation into the feed. However, this process can alter the consistency and perishability of the feed. As a result, producers choose to dilute lipophilic hormones in alcohol, with rapid evaporation, a practice that does not follow the manufacturer’s recommendations (Valle et al 2022).

Given these challenges, the use of nanotechnology can become an alternative for the development of hormonal additives in aquaculture. Among nanocarriers, oil-in-water (O/A) nanoemulsions are surfactant-stabilized colloidal systems in which oil nanodroplets can encapsulate hydrophobic substances such as 17-αMT, promoting controlled release and improving solubility and absorption in biological systems (Elsewedy 2025). In this context, Santos-Magnabosco et  al. (2022) developed a nanoemulsion encapsulating testosterone propionate and demonstrated its effectiveness in inducing sex reversal in tilapia.

Beyond nanoemulsions, other nanostructured systems have also shown significant potential in fish sex reversal. For instance, Yostawonkul et  al. (2023) demonstrated that 17-αMT encapsulated in nanostructured lipid carriers (NLCs) maintained high masculinization efficiency at reduced doses. Similarly, Joshi et  al. (2019) reported that PLGA nanoparticles encapsulating fadrozol, an aromatase inhibitor, achieved successful sex reversal in fish with Page 3 of 19 656 up to a 90% reduction in the required dose. These properties can make it possible to reduce the hormonal concentration used, without compromising its effectiveness, making the sex reversal of tilapia safer, more efficient and sustainable, contributing to the technological advancement of aquaculture.

However, despite these advances, no studies have yet reported the development of oilin-water nanoemulsions containing 17-αMT for oral administration in tilapia feed. This represents a critical research gap, as nanoemulsions may provide a more practical and effective delivery system capable of reducing the hormonal concentration required while maintaining high sex-reversal efficiency. Continued research is needed to develop improved monosex tilapia production strategies, ensuring that aquaculture continues to play a crucial role in the economy and global food security. Thus, the objective of this work was to produce and characterize oil-in-water (O/A) nanoemulsions, containing encapsulated 17α-methyltestosterone, as a hormonal additive incorporated into the commercial feed to sexually reverse fish of the species Oreochromis niloticus and understand the toxicity in the zootechnical parameters of these animals.

Material and methods The experiments were carried out at the Laboratório de Ecofisiologia e Comportamento Animal (LECA) and the Estação de Aquicultura Continental Professor Johei Koike of the Universidade Federal Rural de Pernambuco (UFRPE). Both vivariums are registered on the CIUCA-CONCEA (Brazilian National Council for Animal Experimentation) platform.

All experiments with animals were previously approved by the Ethics Committee on the Use of Animals (CEUA) of UFRPE, protocol number 4288020522.

Material The commercial male hormone 17α-methyltestosterone (17-αMT) for fish farming, in its free form, was purchased from AcquaSupre (São Paulo, Brazil). On the other hand, the male hormone associated with commercial lactose—Alfarever®—composed of 50  g of 17-αMT and 100  g of lactose q.s.p. (50% purity of 17-αMT) was purchased from Nexco (Recife, Brazil) and named 17-αMT + L. The surfactant Tween® 80 (Polysorbate 80, hydrophilic-lipophilic balance = 15.0), nipazol (propylparaben), and absolute ethanol were purchased from Dinâmica (São Paulo, Brazil). The surfactant Span® 80 (sorbitan monooleate 80, hydrophilic-lipophilic balance = 4.3) was purchased from SIGMA (St Luiz, MO, USA). Finally, type 1 soybean oil was used, which, according to normative no.

49/2006 of the Ministry of Agriculture, Livestock and Supply of Brazil (MAPA) (2006), presents the highest quality standard in acidity, purity, and moisture. All other reagents used in the research were of analytical grade.

Production of nanoemulsions The preparation of the oil-in-water (O/A) nanoemulsions was carried out by the highenergy emulsification method by mechanical stirring as described by Santos-Magnabosco et al (2022), with modifications. Briefly, the preparation of the oil phase was carried out by the homogenization of 7.5–12.5% (w/w) of soybean oil and 7.5–12.5% (w/w) of Span 80 for 5 min at a relative centrifugal force of 7 g. To incorporate the hormone, 0.6% (w/w) of 656 Page 4 of 19 17-αMT was previously dissolved in 1 mL of absolute ethanol. Ethanol was used as a solvent because 17-αMT is highly hydrophobic and poorly dispersible in oil alone; dissolving it in ethanol ensured complete solubilization and uniform distribution within the oil phase before emulsification. This mixture remained in mechanical stirring for another 5 min at a relative centrifugal force of 7 g for complete homogenization and evaporation of ethanol.

Simultaneously, the aqueous phase was prepared by homogenizing 69% (w/w) water and 7.5–12.5% (w/w) of Tween 80, remaining in stirring for 5 min at a relative centrifugal force of 7 g. Then, also to ensure complete solubilization and uniform distribution, 0.4% (w/w) of propylparaben diluted in 1 mL of absolute ethanol was added for another 5 min of stirring at a relative centrifugal force of 7 g, also for complete homogenization and evaporation of the ethanol. After preparation of the two phases, the oily phase was dripped into the aqueous phase and submitted to constant agitation at a relative centrifugal force of 60 g for 24 h to obtain the nanometric size of the vesicles.

Experimental design A complete 2 experimental design, with triplicate of the central point (Table 1), was performed in random order (Cadena et al. 2013). Eleven formulations were prepared containing different weights of the components of the oily (O) and aqueous (A) phases (Table 2).

These formulations were used to study the effect of three independent variables being soybean oil, Span 80, and Tween 80. The physicochemical properties of the formulations, such Table 1 Complete 2

experimental design with the levels of the factors (contrast coefficients − 1, 0 and + 1) for the dependent variables used for the production of nanoemulsions FactorsLevels Low (− 1)Central (0)High (+ 1) Independent variables Soybean oil (%)7.510.012.5 Span 80 (%)7.510.012.5 Tween 80 (%)7.510.012.5 Table 2 Study of the effect of three experimental factors (soybean oil, Tween 80, Span 80) by complete 2

experimental design with triplicate of the center point. 17-αMT 17-alpha-methyltestosterone FormulationWater (%)Soybean oil (%) Tween 80 (%) Span 80 (%) Testosterone 17-αMT (%) Propylparaben (%) Total (%) F169.07.57.57.50.60.4100.0 F269.012.57.57.50.60.4100.0 F369.07.512.57.50.60.4100.0 F469.012.512.57.50.60.4100.0 F569.07.57.512.50.60.4100.0 F669.012.57.512.50.60.4100.0 F769.07.512.512.50.60.4100.0 F869.012.512.512.50.60.4100.0 F9*69.010.010.010.00.60.4100.0 Page 5 of 19 656 as vesicle size (nm), polydispersity index (PDI), and zeta potential (mV), were considered dependent variables. In addition, the value of the hydrophilic-lipophilic balance (HLB) was calculated with the following equation (Chong et al 2018):

where HLB mix is the value of the surfactant mixture and HLB and HLB are the HLB values of Tween 80 and Span 80, respectively, while F and F are the values of the fraction by weight of Tween 80 and Span 80, respectively, in the mixture.

Stability studies Preliminary stability tests The formulations produced were evaluated soon after preparation, and again, after centrifugation, cooling, and freezing thermal stress tests to identify potential instability in the production process. Physical and organoleptic parameters, such as dispersion phase, consistency, opacity, color, odor, and lump formation (ANVISA 2004; Santos-Magnabosco et al 2022), were used to evaluate the formulations produced.

Centrifugation test Soon after evaluation of the physical and organoleptic parameters (“Preliminary stability tests” section), the formulations were submitted to centrifugation stability tests, where aliquots of the formulations were packed in 2/3 of the volume of the Eppendorf’s, which allows gas exchange, and were subjected to agitation for 30 min at 1210 g. Next, the preliminary stability was again evaluated (“Preliminary stability tests” section).

Freeze–thaw cycle In order to evaluate possible refrigerated transport, the formulations were also subjected to 24-h thermal stress by cooling at 5 ± 2 °C and freezing at − 20 ± 2 °C. The organoleptic parameters (“Preliminary stability tests” section) were evaluated again immediately after removal from the refrigerator and freezer and immediately after resumption of the formulations at room temperature (25 ± 2 °C).

Physicochemical characterization The physicochemical evaluation of the nanoemulsions was performed by the standard photon correlation spectroscopy (PCS) technique fixed at 90° at 25 °C in a Zetasizer Nano ZS (Malvern, UK). Twenty microliters of the formulations were diluted in 980 μL of doubledistilled water, in order to reduce turbidity, and were analyzed in triplicate for analysis of the mean vesicle size (nm), polydispersity index (PDI), and zeta potential (mV) (Cadena et al. 2013; Santos-Magnabosco et al 2022).

HLB

mix 

HLB

∗F  + 

HLB

∗F  656 Page 6 of 19 Production of hormonal additives and incorporation into commercial feed The formulation with the best organoleptic and physicochemical parameters was chosen as the hormonal additive and was incorporated into the pelleted commercial feed (AcquaPrime®, 45% crude protein). For this, initially, the formulation was dissolved in distilled water and, later, the mixture was added to the previously macerated commercial diet. The proportions used were 100:1:10 or 100:0.5:10 of feed, nanoemulsion, and distilled water, respectively, depending on the experimental group, to obtain 60 mg and 30 mg of 17-αMT per kilogram of feed (Table 3). The commercial hormone 17-αMT + L was used as a positive control. To prepare the mixture, 6 mg of the product was diluted in type 1 soybean oil and mixed with 100 g of diet, following the manufacturer’s recommendations for obtaining 30 mg of 17-αMT per kilogram of diet (Table 3).

All diets containing hormonal additives were manually homogenized with glass rods in beaker, prepared weekly, and stored in a refrigerated environment at 5 ± 2 °C and protected against light.

Experimental fish Tilapia (Oreochromis niloticus) larvae were acquired from a commercial fish farm at Fazenda Campos, in the municipality of São José da Laje in Alagoas state, Brazil. Upon arrival at the vivarium of UFRPE, they were acclimatized for 5 days in glass aquariums with photoperiod (light/dark) 14/10  h, temperature of 27 ± 1  °C, and pH of 7.0 ± 0.5, until complete yolk sac absorption (approximately 7 days after fertilization) and health monitoring.

The experiments were divided into three periods (Matos et  al. 2022), i.e., (i) sex reversal; (ii) rearing; and (iii) fattening, according to the experimental design in Fig. 1.

In all, 720 animals were used, divided equally into four experimental groups (4 × 180 animals per group) described in Table 3. The physicochemical parameters of the water were monitored for optimal levels for the species, such as temperature of 27 ± 1 °C, photoperiod 14/10  h (light/dark), pH of 7.0 ± 0.5, ammonia, nitrite and nitrate with levels below 0.25 ppm, and dissolved oxygen maintained > 6.0 mg/L by artificial aeration. The water was partially renewed two to three times a week, and constant cleaning through siphoning, which prevents the accumulation of organic matter.

Table 3 Details of the experimental groups used in the sex reversal tests of tilapia 17-αMT 17α-methyltestosterone, L lactose.

GroupDescription of the experimental group Control (CON)Fed with powdered feed without additives Commercial hormone (17-αMT + L)Fed with powdered feed added with commercial hormone with a concentration of 30 mg of 17-αMT associated with lactose (17- αMT + L) per kilogram of feed Nanoemulsion 30 mg (NE30)Fed with powdered feed added to nanoemulsion with a concentration of 30 mg of 17-αMT per kilogram of feed Nanoemulsion 60 mg (NE60)Fed with powdered feed added with nanoemulsion with a concentration of 60 mg of 17-αMT per kilogram of feed Page 7 of 19 656 Sex reversal First, each experimental group was divided into six 10-L acrylic aquariums, containing 30 fish each, totaling 180 animals per experimental group, for 28 days for sex reversal and 2  days for zootechnical parameters analysis (Fig. 1). Feeding occurred six times a day with a 2-h interval between each feeding, with the diet containing the hormonal additives as shown in Table 3, to induce sex reversal.

Initially, the diets were sprayed and offered in the proportion corresponding to 30% (w/w) of the live weight of the animals. Subsequently, the rations began to be administered ad libitum adjusting to the satiety of the animals in a standardized way. At the end of the 28 days of sex reversal, all animals were fed with powdered feed without hormonal additives. The measurement of the zootechnical parameters (“Zootechnical parameters and survival” section) of each experimental group, such as fish size and weight, was conducted on the following 2 days. Survival in this period was verified daily (“Zootechnical parameters and survival” section).

Rearing After 30  days of experiment (Fig. 1), the rearing period began. The fish were redistributed equally in five reservoirs of 100 L per experimental group to improve growth conditions, reducing the population density per area, becoming 0.35 fish per liter being ideal for the species (Maeda et al 2010). The feed remained ad libitum, six times a day with sprayed commercial feed without hormonal additives as described above. The feed started to be macerated manually in the mortar with a pistil adjusting the granulometry according to the growth of the fish. This period lasted 83 days. The measurement of the zootechnical parameters of each experimental group and survival of this period were also evaluated (“Zootechnical parameters and survival” section).

Fig. 1 Timeline of the experiment with tilapia, with day 1 being the beginning of feeding for sexual reversal and day 186 being the last day of the study where euthanasia was performed

656 Page 8 of 19 Fattening After 113  days of experiment (Fig. 1), after rearing, the fish were transferred to the Professor Johei Koike Continental Aquaculture Station of UFRPE, where the fattening phase began. In this phase of the experiments, the fish were stored in two net tanks per group, with dimensions of 1.0 m × 1.0 m × 0.7 m (width, length, and depth, respectively), in a semi-excavated pond with 250 m and an average depth of 1  m. Feeding started to be administered four times a day, ad libitum, for a period of 76 days with the same commercial ration without the presence of hormonal additives. The zootechnical parameters of each experimental group and survival of this period were also measured (“Zootechnical parameters and survival” section).

Endpoints evaluated Zootechnical parameters and survival The zootechnical parameters of the fish, such as length and weight, for the evaluation of the toxicity of the hormonal additives were evaluated at the end of each experimental period: (i) sex reversal, (ii) rearing, and (iii) fattening. During the period of sex reversal and rearing, length measurements were performed by analyzing dorsal view images of the animals, captured while kept elongated. The measurements were processed with the ImageJ software (version 1.54d, National Institutes of Health, MD), considering the distance from the anterior portion of the mouth to the posterior end of the tail. For weight, a previously tarted aquarium was used, in which the fish were added, recording the total weight. At the end of the fattening period, there was euthanasia. Then, the length was measured with an analytical caliper, also from the anterior end of the mouth to the posterior end of the tail. To measure weight in this period, the animals were placed on a precision analytical scale and weighed (Acar et  al 2015). The survival percentage was calculated for each experimental period (reversal, rearing, and fattening), considering the initial number of individuals and the losses observed by the period.

Euthanasia, collection of gonads and percentage of reverted animals.

After the fattening phase, the animals were euthanized by associating anesthetic overdose with hypothermia. Briefly, the fish were immersed in water with anesthetic alcohol solution (eugenol 300 mg/L) (Vidal et al 2008) at low temperature (0 °C). After euthanasia and measurement of zootechnical parameters, the gonads were collected and the sex of each individual was identified, based on the investigation of the coelomic cavity after ventral incision in the animals, with the aid of surgical instruments (Matter et  al 2024). Testis were recognized as paired, elongated, whitish to cream-colored organs with a smooth surface and firm consistency, whereas ovaries were identified as paired, lobulated, translucent to yellowish organs, often containing visible oocytes and exhibiting a softer and granular texture (Muazu et al 2020; Paankhao et al 2025). No ambiguous or intermediate gonads were observed during macroscopic examination.

Page 9 of 19 656 Statistical analysis The complete 2 experimental design was analyzed by one-way ANOVA with the Statistica v. 14 software (TIBCO, USA), with the data represented by the Pareto chart.

In the experiments with animals, each individual was considered a sample unit for the statistical tests, and the zootechnical parameters were analyzed as follows:

Weight gain rate (WGR%) (Sherif et al 2024):

Specific growth rate (SGR %/d) (Sherif et al 2024):

Survival (S %) (Siddik et al. 2014):

The results were organized and analyzed in three test periods, with sex reversal, rearing, and fattening being analyzed independently (Sarker et al. 2022). These results were expressed as mean and standard deviation, and the statistical analysis was performed by one-way ANOVA using the Origin Pro Academic 2015 software (Origin Lab. Northampton, MA, USA). When the difference was significant, the means were compared using Tukey’s test with p < 0.05.

WRG=100×

finalbodyweight−initialbodyweight initialbodyweight

SGR=100×

In(finalbodyweight)−ln(initialbodyweight) days S=100× finalnumberoffish initialnumberoffish Table 4 Evaluation of the organoleptic parameters, according to Brazil (2004), of oil-in-water (O/A) nanoemulsions in relation to the variations of their constituents such as Tween 80, Spam 80, and soybean oil, after 24 h of mechanical agitation FormulationAspect (dispersion phase)Aspect (consistency)Appearance (opacity) ColorOdorLumps F1HomogeneousFluidOpaqueAbsentAbsentAbsent F2HomogeneousFluidOpaqueAbsentAbsentAbsent F3HomogeneousFluidOpaqueAbsentAbsentAbsent F4HomogeneousFluidOpaqueAbsentAbsentAbsent F5HomogeneousNot very thickOpaqueAbsentAbsentAbsent F6HomogeneousFluidOpaqueAbsentAbsentAbsent F7HomogeneousFluidOpaqueAbsentAbsentAbsent F8HomogeneousFluidOpaqueAbsentAbsentAbsent F9*HomogeneousFluidOpaqueAbsentAbsentAbsent 656 Page 10 of 19 Results Stability assessment Preliminary stability test At the end of the preparation of the formulations, they all presented the same characteristics regarding the dispersion phase, opacity, color, odor, and presence of lumps (Table 4); they were homogeneous, opaque, and without color, odor, and lumps. Regarding fluidity (Table 4), most of the formulations were fluid, except for F5, with a thin consistency, which had a higher amount of Span 80 (12.5%) and lower amounts of oil and Tween 80 (both with 7.5%).

Centrifugation test The formulations were submitted to the centrifugation test at 1210  g for 30  min, followed by their preparation (Table 5). With the exception of F5 and the central point (F9), all the others were observed phase separation, characterized as heterogeneous.

Freeze–thaw cycle.

During the cooling process (Table 5), the formulations F1, F2, F3, F4, and F8 showed phase separation. Regarding freezing (Table 5), with the exception of the F1 formulation, all the others remained stable when frozen and when returning to room temperature (25 °C).

Table 5 Effect of the variation in the amount of constituents such as Tween 80, Spam 80, and soybean oil of oil-in-water (O/A) nanoemulsions containing 17-alpha-methyltestosterone in relation to the dispersion phase (aspect: homogeneous or heterogeneous) after centrifugation process at 1210 g and Freeze–thaw cycle, cooling at 5 ± 2 °C and freezing at − 20 ± 2 °C FormulationCentrifugation (1210 g/30 min) Cooling (5 ± 2 °C)Freezing (− 20 ± 2 °C) F1HeterogeneousHeterogeneousHeterogeneous F2HeterogeneousHeterogeneousHomogeneous F3HeterogeneousHeterogeneousHomogeneous F4HeterogeneousHeterogeneousHomogeneous F5HomogeneousHomogeneousHomogeneous F6HeterogeneousHomogeneousHomogeneous F7HeterogeneousHomogeneousHomogeneous F8HeterogeneousHeterogeneousHomogeneous F9*HomogeneousHomogeneousHomogeneous Page 11 of 19 656 Physicochemical analysis The results of the physicochemical analyses of the formulations are presented in Table 6.

All formulations presented pH 8.0. Although all the vesicles were on a nanometric scale, there is a difference in size between the formulations. The largest sizes were found from F1 to F4 formulations, with a size between 203.40 and 374.70  nm. In addition, the smallest sizes were found from F5 to F9 formulations, with a size between 97.73 and 102.21  nm (Table 6). The size of the vesicles is inversely related to the amount of span 80, as seen in the Pareto chart (Fig. 2A). The formulations with the lowest amounts of span 80 (7.5%) were the ones with the largest vesicle size. In addition, the vesicles with the smallest sizes were found in formulations F5 to F9, which had the highest amounts of Span 80 (Table 6).

Finally, it was also seen that the interaction of two factors, Tween 80 and oil, also had a significant effect on the reduction of vesicle size.

The results of the PDI analysis (Table 6, Fig. 2B) indicated a uniform distribution of vesicle size (< 0.7), according to Danaei et  al. (2018). Thus, in the analysis of the independent variables, no significant effect was observed on the PDI (Fig. 2B).

All formulations showed negative Zeta potential, ranging from − 27.10 to − 43.10  mV (Table 6). The Pareto chart in Fig. 2C revealed that the interaction between oil and Tween 80 caused a negative effect on the zeta potential, showing that the choice of the amount of oil and surfactant, respectively, is important in obtaining the vesicle load.

Based on the results obtained, the formulation selected for incorporation into the diet was the central point (F9), which contained a lower proportion of Span 80, a surfactant of high commercial value, while still providing excellent stability. F9 remained stable in terms of organoleptic and physicochemical parameters, showed nanometric vesicle size, presented homogeneous PDI values (< 0.300), and exhibited a zeta potential higher than − 30 mV, all of which indicate good colloidal stability.

Zootechnical parameters and survival Measurements of the zootechnical parameters of the fish were performed at the beginning and end of each period of the experiment (sex reversal, rearing, and fattening) to evaluate toxicity, and the results are presented in Table 7. After sex reversal, all groups fed hormonal additives showed similar results for weight gain rate (WGR) and specific Table 6 Evaluation of the hydrophilic-lipophilic equilibrium (HLB) values and physicochemical parameters of the oil/water (O/A) 17-alpha-methyltestosterone nanoemulsions in relation to the variations of its constituents such as Tween 80, Span 80, and soybean oil FormulationHLBpHSize (nm)PDIZeta (mV) F19.658.0267.20 ± 13.650.159 − 42.10 ± 1.50 F29.658.0285.90 ± 20.770.278 − 36.30 ± 0.21 F310.998.0374.70 ± 38.340.571 − 33.70 ± 1.24 F410.998.0203.40 ± 7.320.253 − 27.10 ± 0.93 F58.318.098.01 ± 2.150.284 − 43.10 ± 1.04 F68.318.0102.70 ± 2.590.247 − 33.00 ± 1.72 F79.658.0153.40 ± 0.230.247 − 38.30 ± 1.62 F89.658.097.73 ± 0.820.137 − 34.70 ± 1.50 F9*9.658.0102.21 ± 30.350.287 − 38.20 ± 1.56 656 Page 12 of 19 Fig. 2 Pareto chart evaluating the influence of the factors span 80, tween 80, soybean oil and their combinations for the response variables A vesicle size (nm), B polydispersity index (PDI), and C Zeta potential (mV)

Page 13 of 19 656 growth rate (SGR) and final weight. Also, there was no significant difference in the final size of the fish between the experimental groups. At the end of the rearing period, the groups fed with hormonal additives showed a significant difference between the final weight compared to the control group. After the fattening period, the animals presented zootechnical parameters similar to the control group.

Survival results are also shown in Table 7. It was observed that all experimental groups had mortality values below 30% during all periods of the experiment, values that are within the expected for sex reversal and age of the animals (Fessehaye et  al 2006;

Zaki et al 2021). Based on these results, this may indicate that the hormonal additives did not show toxicity and are safe for use in sex reversal.

Table 7 Zootechnical paramters and survival of experimental groups of tilapias after sex reversal. CON control group, 17-αMT + L group with commercial additive, NE30 nanoemulsion group with 30 mg of testosterone, NE60 nanoemulsion group with 60 mg of testosterone *Statistically significant difference in relation to the control (p < 0.05) by Tukey’s test

CON17-αMT + LNE30NE60

Sex reversal Starting size (cm) 1.130 ± 0.1541.130 ± 0.1541.130 ± 0.1541.130 ± 0.154 Final size (cm)2.548 ± 0.3302.711 ± 1.5652.530 ± 0.4072.540 ± 0.420 Starting weight0.017 ± 0.0010.017 ± 0.0010.017 ± 0.0010.017 ± 0.001 Final weight0.246 ± 0.0930.264 ± 0.1260.265 ± 0.1340.281 ± 0.155 SGR (%)8.550 ± 1.2608.610 ± 1.7208.600 ± 1.7208.790 ± 1.710 WGR (%)1297.034 ± 528.2001398.260 ± 718.7301405.010 ± 761.9701492.760 ± 879.370 Survival in the period (%) 85.081.177.281.1 Rearing Starting size (cm) 2.548 ± 0.3302.711 ± 1.5652.530 ± 0.4072.540 ± 0.420 Final size (cm)10.799 ± 0.73010.667 ± 0.92811.185 ± 0.67310.914 ± 0.656 Starting weight0.246 ± 0.0930.264 ± 0.1260.265 ± 0.1340.281 ± 0.155 Final weight17.712 ± 1.64618.956 ± 4.915*19.449 ± 2.730*19.419 ± 2.598* SGR (%)5.144 ± 0.1115.113 ± 0.2755.160 ± 0.1725.092 ± 0.156 WGR (%)7081.214 ± 667.4357065.999 ± 1858.2937219.393 ± 1027.5216805.604 ± 924.008 Survival in the period (%) 96.786.398.693.2 Fattening Starting size (cm) 10.799 ± 0.73010.667 ± 0.92811.185 ± 0.67310.914 ± 0.656 Final size (cm)17.410 ± 2.22218.697 ± 2.31618.199 ± 2.06018.036 ± 1.713 Starting weight17.712 ± 1.64618.956 ± 4.91519.449 ± 2.73019.419 ± 2.598 Final weight95.657 ± 32.864106.260 ± 37.639101.790 ± 31.996103.385 ± 30.259 SGR (%)2.051 ± 0.4772.071 ± 0.6072.011 ± 0.5112.058 ± 0.398 WGR (%)440.050 ± 185.538460.564 ± 198.562423.369 ± 164.512432.383 ± 155.821 Survival in the period (%) 98.095.298.597.8 656 Page 14 of 19 Evaluation of sex reversal in tilapia The results of sex reversal are presented in Table 8. At the end of 189 days of the experiment, the 17-αMT + L group, used as a reference, obtained sex reversal efficiency > 99%, showing satisfactory performance of the product with 50% (30  mg/kg of 17-αMT) of the concentration of the hormone commonly used in sex reversal. The high efficiency (> 99%) of sex reversal was also observed in the NE30 and NE60 groups, with the concentration of 30 mg/kg (NE30) being the most indicated, due to the reduction of the necessary concentration of hormone to obtain the desired sex reversal efficiency. Finally, hormonal additives developed in this study obtained the same level of efficiency as the product on the market.

Discussion The effectiveness of nanoemulsion production process is directly related to the stability of the colloidal system, and it is essential that the vesicles remain uniformly dispersed and resistant to coalescence over time and under variable environmental conditions (Devi et al 2025; Elsewedy 2025). In the present study, although all formulations showed initial homogeneity, only F5 and F9 maintained stability after centrifugation and thermal stress tests. The F5 formulation, characterized by a lower proportion of oil and Tween 80 and a higher proportion of Span 80, had a thin consistency, which can be attributed to the lipophilic character of Span 80, which favors the formation of a dense interfacial barrier, making it difficult for vesicle coalescence (Chong 2018; La Cruz 2022). While in the F9 formulation, the central point of the experimental design, it presented equal proportions of oil, Tween 80, and Span 80, and demonstrated good fluidity and colloidal stability. The stability observed in F5 and F9 also reinforced that the different proportions of the constituents of the nanoemulsions can be effective, if they are adequately balanced. Consistently, the experimental design confirmed that adjusting surfactant proportions was essential for achieving stability (Tables 4, 5, and 6), a finding in line with Iskandar et  al. (2024), who optimized four groups of nanoemulsions with different combinations of surfactants, including the Span 80 and Tween 80 duo, resulting in stable systems, with high encapsulation efficiency and antioxidant activity.

Building on these results, analysis of independent variables highlighted Span 80 as a key factor in reducing vesicle size, maintaining size uniformity, and achieving high zeta potential, all indicative of good colloidal stability (Devi 2025). This was also observed by Table 8 Gender distribution of the groups studied, detailing the number of males and females, as well as the corresponding percentage in relation to the total of each group. CON control group, 17-αMT + L group with commercial hormone, NE30 nanoemulsion group with 30 mg of hormone per kilogram of feed, NE60 nanoemulsion group with 60 mg of hormone per kilogram of feed GroupTotal animalsNo. of malesNo. of females% of males% of females

CON145955065.52%34.48%

17-αMT + L120119199.17%0.83%

NE30135134199.26%0.74% NE60133125893.98%6.02% Page 15 of 19 656 Faghmous et al. (2020), where the authors observed that increasing Span 80 led to reduced vesicle size, while intermediate concentrations of Tween 80 were associated with maintaining this reduction and increasing zeta potential. Additionally, Shahavi et  al. (2019) also highlighted the role of Span 80 in the stabilization of oily nanoemulsions, especially when combined with hydrophilic surfactants, such as Tween 80. In addition, a limitation of our study is that encapsulation testosterone efficiency was not determined. Future work may obtain this data.

The synergistic interaction between Span 80 and Tween 80 has been widely described as an effective strategy for the formation of stable colloidal systems. For example, Chong et al. (2018) observed that the combination of Span 80 and Tween 80 provided high stability than the use of Tween 80 alone, promoting reinforcement of the interfacial film and significant reduction of coalescence. Similarly, Mahmud et  al. (2024) demonstrated that mixing these surfactants with HLB adjusted to 12, using optimized ratios, resulted in nanoemulsions with reduced average size (38.11 nm), low polydispersity index (0.27), and high zeta potential (+ 37.8  mV), desirable parameters for physicochemical stability. The efficacy of this combination can be explained by the complementarity of their HLB values:

Span 80 (HLB 4.3), more lipophilic, contributed to stabilizing the oil phase, while Tween 80 (HLB 15), more hydrophilic, acted to stabilize the aqueous interface (Chong et al 2018;

Shahavi et al 2019; La Cruz et al 2022). The mixture of these surfactants allows for the formation of a more effective interfacial barrier, promoting the reduction of interfacial tension and the prevention of coalescence (Iskandar et  al 2024). This occurred in the production process of our formulations, which may justify the good values in the organoleptic and physicochemical parameters found in our study.

In the present study, the HLB calculated for the F5 formulation was 8.31 and for the F9 formulation it was 9.65. The choice of F9 as the ideal formulation is justified by the fact that its HLB is closer to the values described in the literature as ideal for oil-in-water (O/A) emulsions, which generally vary between 8 and 16, depending on the oil used (Chong 2018; Shahavi 2019; Mahmud 2024). For example, La Cruz et al. (2022) reported that mixing Tween 80 and Span 80 with HLB 10.46 resulted in an emulsion with visual characteristics typical of a nanoemulsion, while Shahavi et al. (2019) produced a stable nanoemulsion that remained unchanged for 6 months using the same surfactant combination with an HLB of 9, taking into account ultrasonication parameters, oil/surfactant ratio, and the type of essential oil (clove oil).

The results of our study demonstrated that nanoemulsions with testosterone encapsulated were an efficient and safe alternative for the sex reversal of tilapia, without significantly compromising the zootechnical parameters of the fish. The results available in the literature showed variations in the impact of 17α-MT on zootechnical parameters, which can be attributed to other factors such as stocking density, environmental conditions, and administration methods (Yostawonkul et al 2023; El-Greisy 2012; Costa e Silva et al. 2022;

Ramirez 2024), which was not seen in our study.

Regarding survival, the results of our study indicated that hormonal treatments did not compromise the viability of the fish, with mortality rates below 30% in all periods. This is in line with the expected values for sex reversal in tilapia farming (Fessehaye et  al 2006;

Zaki et al 2021; Asad et al 2023). Nevertheless, considering the risks associated with exposure to androgenic hormones, such as impacts on fish health and environmental contamination (Abo-Al-Ela 2018; Hasheesh et al 2011; Andersen et al 2006), there is a tendency to reduce the hormonal concentration used in sex reversal processes, while maintaining efficacy. Currently, the conventional method of sex reversal uses 60 mg of 17α-MT per kg of feed for 28 days (Sarker et al 2022). However, in our study, it was possible to achieve a 656 Page 16 of 19 sex reversal rate higher than 99% using a 50% lower concentration. This was also achieved in our study with the reduction from 60 to 30 mg per kg of feed.

This performance surpasses that reported in several previous studies. Amer et  al.

(2021), for example, offering 30 mg of 17α-MT per kg of feed obtained 70% sex reversal, while offering 60 mg/kg obtained 90%, for a period of 28 days. Jensi et al (2016) obtained 83.3% reversal with 50 mg/kg and 93.3% with 60 mg/kg after 21 days of treatment. Similar results were reported by Sarker et al. (2022) where they obtained 86.1% with 50 mg/kg and 94.4% with 60 mg/kg for 28 days, while El-Greisy (2012) obtained 88% with 40 mg/kg and 95% with 60 mg/kg. These results reinforce that the hormonal nanotechnological additive presented in our study is among the best available with industrial viability. This also demonstrates the need to develop technologies capable of reducing the hormone concentration applied in production systems, maintaining or even increasing the efficiency of the process.

In this context, encapsulation systems such as nanoemulsions and nanoparticles emerge as alternatives for tilapia farming. The nanoemulsions encapsulate the hormone in a lipid matrix and then have the potential to control the release of hormones into the biological system. This can reduce side effects and improve absorption, as well as minimize the risk of environmental contamination. Similar results have been observed in literature with the use of nanotechnological strategies. Santos-Magnabosco et  al (2022) reported a sex reversal rate of 75% with testosterone propionate nanoemulsion after 28 days. Joshi et al.

(2019) used PLGA nanoparticles encapsulating fadrozole and reduced the concentration from 500 to 50  ppm, maintaining sex reversal rates between 95 and 97% in 15  days, and reaching 100% in 30 days. Similarly, Yostawonkul et al. (2023) developed a nanostructured lipid transporter containing nanoencapsulated methyltestosterone in polyglycoside aquil, reducing the concentration from 60 to 30 ppm in a 21-day experiment, with a reversal efficiency of 98.5%. Our results were similar in terms of efficiency (> 99%) and reversal time (28 days) to the other hormonal nanocarriers described above. Such results, together with those obtained in this study, show the potential of hormone nanocarriers as an effective, highly efficient, environmentally safer tool, with a reduction in the time of hormone use and concentration for sex reversal in tilapia farming, allowing the development of more competitive marketing products.

Conclusion This study demonstrated the successful production, characterization, and in  vivo evaluation of oil-in-water (O/A) nanoemulsions containing encapsulated 17α-methyltestosterone for tilapia sex reversal. Nanoemulsions containing 10% soybean oil, 10% Tween 80, and 10% Span 80 showed better physical–chemical stability under different conditions, maintaining their homogeneity, obtaining uniform nanometric vehicles with surface charges of − 38  mV. Additionally, in controlled trials, these nanoemulsions allowed a 50% reduction in the hormonal dose required for sex reversal, achieving a masculinization rate of 99% with 30  mg of 17α-MT per kg of feed, while maintaining survival rates above 70%.

Finally, they allowed better solubility of the food additive, eliminating the need for dilution in oil or alcohol. Therefore, 17α-methyltestosterone encapsulated in nanoemulsions represented an efficient and safe solution for the sex reversal of tilapia, with the potential to improve the sustainability and efficiency of aquaculture.

One of the limitations of this study was the standardized time for sex reversal (28 days).

Future works will focus in reduce the time for sex reversal and the optimization of Page 17 of 19 656 testosterone concentrations studying these factors and their interactions by experimental designs. Testosterone in the environment is an endocrine disruptor, and future works will need to reduce the use of hormones on aquaculture.

Author contributions Details of each author with their contribution in this paper: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Software, Validation, Visualization, Writing - original draft, Writing - review & editing were performed by Matheus Victor Viana de Melo, Amanda Rodrigues dos Santos-Magnabosco, Jadson Freitas da Silva, Aline Rafaely Damaso Alves, Hansheys Ménard, Stefanny Arielle Gomes da Silva, Dijaci Araújo Ferreira, and Pabyton Gonçalves Cadena **.** Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing - original draft, Writing - review & editing were performed by Pabyton G. Cadena. All authors read and approved of the final manuscript.

Funding This work was supported by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) for scholarships to graduate students, CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) for the productivity grant to Pabyton Cadena (302399/2023–2).

Data availability The data and materials used in this study are available from the corresponding author on request.

Declarations Ethics approval The protocols used in this study were approved by the Ethics Committee in the use of animals of the Universidade Federal Rural de Pernambuco—Brazil, License number license 4288020522. All experiments followed national and international guidelines. No human participants were involved in this article’s studies.

Competing interests The authors declare no competing interests.

Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

[conteúdo visual do documento] [página 5] Fig. 2 — Imagem de peixe com marcações em vermelho:

TL HH ED

Fig. 1 — Diagrama “METHODOLOGY OF THE SEX REVERSAL EXPERIMENT” / “FISH DEVELOPMENT STAGES”:

0 DPF

Start of fed with nanoemulsion-added feed 6 times a day for 30 days.

14 DPF

START OF SEX REVERSAL

12–13 DPF

COMPLETE ABSORPTION OF YOLK SAC

The animal begins to ingest food. This is the ideal period to start introducing the feed with testosterone as sexual differentiation has not yet started.

(FUJIMURA/OKADA 2007, POPMA/LOVSHIN 1996)

17 DPF

START OF DEFINITION OF PHENOTYPIC SEX

During this period, ovotestis begins to differentiate into testis or ovary, as this is when phenotypic sex is fixed.

(TAO et al. 2018)

32 DPF

END OF DEFINITION OF PHENOTYPIC SEX

42 DPF

END OF SEX REVERSAL

42 DPF

START OF THE SEXUAL DIFFERENTIATION

The increase of expression of 2 genes, Dmrt1 gene [testis] and Cyp19a1a gene [ovary], responsible to sex differentiation. The morphological differences begin to become visible.

(TAO et al. 2018)

47 DPF

EUTANASIA AND MORPHOMETRY

52 DPF

END OF THE SEXUAL DIFFERENTIATION

The animals stop eating nanoemulsion-added feed and start eating the feed without hormone.

[página 10] Fig. 3 — Fotomicrografias com painéis:

A B C D

Marcadores visuais presentes nos painéis: setas/triângulos pretos e estrelas pretas indicando células germinativas.

[página 15] Elemento visual lateral:

Check for updates

Rodapé:

Published online: 15 November 2025

[página 21] Fig. 1 — Linha do tempo do experimento:

Day 01 Sex reversal

End of sex reversal Day 28

Day 30 Juvenile rearing

Fattening Day 113

Day 189 Euthanasia, Morphometry and Gonad Collection.

[página 26] Fig. 2 — Pareto chart.

Painel A:

A (3)Span 80 (g): -7.60033 1by2: 2.80211 (1)Oil (g): -2.27647 1by3: 1.137372 (2)Tween 80 (g): .8431473 2by3: .2852754 p=.05

Painel B:

B 2by3: -1.65969 1by2: -1.5851 (3)Span 80 (g): -1.07538 (1)Oil (g): -1.07538 (2)Tween 80 (g): .739713 1by3: .1595459 p=.05

Painel C:

C (1)Oil (g): 7.500675 (2)Tween 80 (g): 5.985194 2by3: -4.18196 (3)Span 80 (g): -2.8775 1by2: -1.59221 1by3: .4028496 p=.05

Eixo inferior:

Standardized Effect Estimate (Absolute Value)