Survival, Growth performance and Sex Reversal effect of Hibiscus rosa sinensis leaves ethanolic extract on Nile Tilapia Oreochromis niloticus (Linnaeus, 1758)

MUTLEN Melvin*; ZANGO Paul; FADIMATOU GEREHEH ; NTAKOU PANGO Hervé1 and TOMEDI EYANGO Minette

Department of Aquaculture, Institute of Fisheries and Aquatic Sciences, The University of Douala, Douala,Cameroon;

Received Date: May 26, 2025; Accepted Date: August 16, 2025; Published Date: September 16, 2025

*Corresponding author: MUTLEN Melvin, Institute of Fisheries and Aquatic Sciences, The University of Douala, P0 Box 7236-Douala-Cameroon, E-mail: mmutlen80@gmail.com; Phone numbers: +237694239218 /+237673139110

Citation: Melvin M, Paul1 Z, Gereheh F, Hervé NP, Minette TE [2025] Survival, Growth performance and Sex Reversal effect of Hibiscus rosa sinensis leaves ethanolic extract on Nile Tilapia Oreochromis niloticus (Linnaeus, 1758). Jr Aqua Mar Bio Eco: JAMBE-155.

DOI: 10.37722/JAMBE.2025204

Watch the Article in Motion

Download PDF
Abstract

The present study was conducted to evaluate the effect of Hibiscus rosa-sinensis leaves ethanolic extract on survival, growth performance and masculinization of Oreochromis niloticus fry. To this end, 750 O. niloticus fry with an average weight of 2.69 ± 0. 04 g were randomly distributed in 15 happas measuring 0.7×0.7×1m placed in a 10x 45 m2 earth pound at a density of 50 fry per happa and subjected to natural temperature and light conditions. Offsprings were fed with 5 experimental feeds including 02 controls (negative control (no treatment) and positive control (treatment with 17a – methyltestosterone at 60 mg/kg feed)) and 03 based on H. rosa-sinensis leaves ethanolic extractat doses of 100; 110 and 120 mg/kg feeds. After 60 days post-treatment, survival and zootechnical growth parameters were assessed. The study employed the standard acetocarmine squash technique of gonads to analyze the sex reversal percentage. Phytochemical screening revealed the presence of phenolic compounds, flavonoids, alkaloids, tannins saponins and steroids in H. rosa-sinensis leavesethanolic extract. The result showed that total survival rates in all treatments and controls were ranging from 99.62± 0.64 to 96.66 ±0.67 % (p ˂ 0.05). Fishes from group treated at 0; 110 and 120 mg/kg of H. rosa-sinensis leaves ethanolic extracts obtained the highest survival rates (99.62 ± 0.64%; 99.62 ± 0.64%; 99.44 ± 0.96% respectively). Analysis of the growth parameters revealed that offsprings treated with Methyltestosterone at 60 mg/ Kg feed had a significantly greater effect (p ˂ 0.05) than the other treatments applied in terms of Average Weight Gain (42.54±1.19g), Average Daily Gain (1.01 ±0.02g/d), Specific Growth Rate (6.72±0.45%/d), Average Standard Length (10.43± 1.24cm) and Condition Factor (3.98 ± 0.26(%g/cm3). A comparative analysis of the different group treated with H. rosa-sinensis leaves ethanolic extract (100; 110 and 120 mg/kg) reveals a significant effect of the treatment at the dose of 120 mg/kg on growth performance. A comparative analysis of the sex ratio of treated group with H. rosa sinensis leaves ethanolic extract revealed a significant effect of the treatment at 120 mg/kg with an estimated masculinization rate of 77.77 ± 1.21%. These findings highlight the potential of H. rosa sinensis leaves ethanolic extract as a viable alternative to synthetic steroid hormones for achieving masculinization in Nile tilapia.

Keywords: Nile tilapia Hibiscus rosa sinensis, Sex reversal, Survival, growth performance, 17a-MT

Introduction

      The tilapia Oreochromis niloticus, Linnaeus, 1758, commonly known as “Nile tilapia”, is the most common fish farmed in tropical Africa. A warm-water, farmed fish, it is the mainstay of freshwater fish farming in the world’s intertropical belt [1, 2]. Worldwide, tilapia is the second most farmed and produced group of fish with 3.49 million tonnes (Mt) well after carp (24 Mt), followed by clarids with 2.97 Mt and salmonids with 2.36 Mt [3, 4, 5, 6]. Thanks to its nutritional value, which is rich in essential amino acids and fatty acids of good nutritional quality, tilapia O. nilotocus is very edible, with flesh that is much appreciated by consumers, making it a highly commercialized fish [7, 8]. Rapid growth rates, high tolerance to low water quality, efficient food conversion, resistance to disease, good consumer acceptance and ease of spawning made tilapia a suitable fish for culture [9]. The fish is reported to sexually mature at a small size of around 6 cm and a young age of around 3 months [10]. However, because of its very high reproduction and precocity, tilapia O. niloticus is exposed to frequent cases of dwarfism and close inbreeding. This could have a negative impact on farm production yields [11, 12].

      To circumvent these constraints linked to anarchic reproduction and improve yields by producing high-growth individuals, various methods have been developed to produce all-male tilapias, including manual separation of sexes, hybridization, genetic manipulation, and hormonal sex reversal [13]. Among the methods of producing monosex tilapia, hormonal sex reversal is acknowledged as the most efficient and is a commonly used technique that allows for the mass production of all-male tilapia in both small- and large-scale production systems [14, 15]. Synthetic steroids such as 17-alpha methyltestosterone, androstenedione or 1-dihydrotestosterone acetate are commonly used to induce sex reversal in tilapia, but because of the potential dangers of such steroids, particularly the impact of treatment residues on the health of aquaculture workers, the environment and biodiversity [11, 16], the use of plants with androgenic properties is a potential alternative worth exploring. Herbal extracts are easily accessible, less costly and safe for the environment and humans since they are more biodegradable compared to synthetic hormones [17, 18]. Consequently, plant extracts are considered economically viable and eco-friendly alternatives to synthetic hormones for smallholder tilapia farmers in Africa. Several medicinal herbs have been reported to possess compounds known as phytoandrogens [19] and phytoestrogens [20], which are believed to have functional effects similar to testosterone or estrogens, respectively, in animals. Plant extracts contain phytochemicals capable of inhibiting estrogen biosynthesis and acting as aromatase inhibitors and estrogen receptor antagonists in gonadal germ cells, and can therefore be considered as potential means of sex reversal in fish [21]. Indeed plant extracts contain various bioactive principles such as alkaloids, flavonoids, pigments, phenolics, terpenoids, steroids, essential oils which have been reported to promote various activities such as anti-stress, growth stimulation, appetite stimulation, tonicity and immunostimulant and antimicrobial properties during fish production [22,23]. Reverter et al. [24] and Van Hai [25] indicated that plant extracts containing phytochemicals such as isoflavonoids, flavonoids, and saponins, which possess estrogenic and androgenic properties, could potentially replace synthetic steroid hormones for sex reversal in tilapia.

       Member of the Malvaceae family, Hibiscus Rosa sinensis Linn is a glabrous shrub widely cultivated in the tropics as an ornamental plant with a variety of flower shapes and colours [26]. The various parts of this plant have been reported to have various medicinal properties (hypoglicaemic, antitumour, antioxidant, antihypertensive, antipyretic, anti-inflammatory, analgesic, antibacterial, menstrual cycle regulator, contraceptive, etc.) [27, 28, 29, 30, 31]; this would justify its use by natural health practitioners [27]. Flavonoids, tannins, terpenoids, saponins and alkaloids are the main phytochemical compounds present in various extracts from the leaves, seeds, bark and stem of Hibiscus rosa-sinensis and are most likely responsible for their biological activities [32]. The flavonoids contained in H. rosa-sinensis extracts are thought to induce anti-implantation and anti-spermatogenic effects in animals [33, 34, 35, 36], Several authors have demonstrated the anti-fertility, anti-spermatogenic, anti-oestrogenic and abortive activity of extracts of different parts of the H. rosa-sinensis plant [ 37, 34, 26, 38]. The treatment of Nile tilapia with H. rosa-sinensis leaf powder at 3.0 and 4.0 g /kg of feed ruptured testes and ovary tissues after 60 days of treatment, thereby inducing sterility [39]. The inclusion of H. rosa-sinensis flower extracts also induced masculinization of Nile tilapia fry, producing up to 73.13% male individuals [40]. However, the treatment of Nile Tilapia fry with vary doses of H. rosa-sinensis leaves ethanolic extract (70; 80 and 100 mg/kg of feed) revealed a significant effect of the treatment at 100 mg/kg of feed on growth performance and sex ratio (60.82% of males) [41]. No previous study has attempted to investigate the efficacy of H. rosa-sinensis leaves ethanolic extract on Nile Tilapia masculinization beyond the dose of 100 mg/kg. Hence the interest of this study, which aims to assess the effect of Hibiscus rosa sinensis leaves ethanolic extract on Survival, Growth and Sex Reversal of Oreochromis niloticus (Linn. 1758).

Materials and methods

Experimental Site

      The experimental part was conducted on the production facilities of AQUAKAM production farm located in the city of Yaoundé, Centre Region of Cameroon, with the following geographical coordinates: 3°48‘and 3°51’ north latitude and 11°29‘and 11°35’ east longitude, with an average altitude of 669 m above sea level. The climate in the locality of Soa is sub-equatorial with tropical tendencies, with 4 seasons: 2 dry seasons from December to January and July to August; 2 rainy seasons from October to November and March to April. The temperature ranges from 24.9°C to 28.2°C, with an average of 27.5°C [42].

Acquisition of Orechromis niloticus hatchling

      The broodstock used in these experiments consisted of Sixty sexually mature Nile Tilapia (15 males and 45 females, about 7–9 months old; average weight ± SE = 64.88 ± 1.22 g) from the initial stock of the Aquakamfarm. They were stocked (at a ratio of 1 male: 3 females) in a triple happas of 1.5 m³ each, placed in an 80m² ponds. The broodstock were fed 3 times a day (with a pelleted feed of 30% CP) and monitored for spawning during 40 days. From the 30th day, freshly hatched O. niloticus fry was collected from the happa and distributed randomly in 15 happas at a rate of 50 fry / happa according to the treatment. They were subjected to natural conditions of temperature and photoperiod. Larvae at different stages could be harvested, but the ones of interest for this experiment were those that still had a yolk sac, to prevent them from ingesting exogenous food.

Collection of plant material

      2,5kg of fresh Hibiscus rosa-sinensis leaves were harvested in their natural habitat near the Aquakam production farm located in the city of Yaoundé-Cameroon. The collected plant material was cleaned and then dried for 14 days away from the sun, as drying in the sun could cause photoreactions that could alter the molecules of certain active ingredients [43]. The leaves were dried under shade and milled into a fine powder; the powder (236 g) was kept in a dry, clean, airtight plastic container at room temperature until usage.

Preparation of Hibiscus rosa-sinensis leaves ethanolic extracts

      236 g of Hibiscus rosa-sinensis leaves powder were soaked in 2600 ml ethanol at 95% for 24 hours with constant shaking at intervals as described by Musa et al. [44]. It was filtered using Watmann filter paper, the filtrate was concentrated by drying it in the oven under pressure at a temperature of 45°C for 8 hours. The concentrated extract were stored in clean bottle, labeled and then preserved in the refrigerator until when needed. The yield of evaporated dry extract on the initial weight basis was calculated using the following equation: R (%) = (W1×100)/ W2 where W1: weight of the extract after evaporation of the solvent; W2: dry weight of the initial sample. The yields obtained from the extraction process were 21.44%.

Phytochemical characterisation of Hibiscus rosa-sinensis leaves ethanolic extracts

Phytochemical screening

      Phytochemical screening was carried out on the basis of characteristic color tests to identify the main chemical groups. The various phytochemical groups of Hibiscus rosa-sinensisleavesethanolic extract were characterized using the techniques described by Akrout et al.[45].

Detection of alkaloids (Buchard reaction)

      To 1mL of each solution, 2 drops of Bourchard’s reagent (iodine-iodide reagent) are added. The observation of a reddish-brown precipitate indicates a positive reaction.

Detection of Flavonoids (sodium hydroxide test)

      A few drops of a 10% NaOH solution are added to a tube containing 3 mL of the extract solution. A yellow-orange color indicates the presence of flavonoids.

Detection of Phenolic compounds (reaction with ferric chloride (FeCl3)

      A drop of 2% alcoholic ferric chloride solution is added to 2 mL of extract.  A more or less dark blue-black or green color indicates a positive reaction.

Detection of tannins (Reaction with 1% ferric chloride)

      To 1 ml of extract in a test tube was added 2 ml of water followed by one or two drops of 1% ferric chloride. The appearance of a blue, blue-black or black color indicates the presence of gallic tannins; a green or dark green color indicates the presence of catechic tannins.

Detection of saponins (Foam Index)

      0.1 g of extract was dissolved in a test tube containing 10 mL of distilled water. The tube was shaken vigorously lengthwise for 30-45 seconds and then left to stand for 15 minutes. The height of the foam is measured. The persistence of foam more than 1 cm high indicates the presence of saponins.

Detection of steroids (Salkowski test)

      5 drops of concentrated H2SO4 were added to 1 mL of extract. A red coloration in each extract indicates the presence of steroids.

Quantitative Determination of Phytochemicals

Quantitative Estimation of Alkaloids

      The assay was performed using the spectrophotometric method described by Sreevidya & Mehrotra [46]. A quantity of 5mL of extract solution was taken and the pH was maintained between 2 and 2.5 with dilute HCl. 2mL of Dragendorff’s reagent was added and the precipitate formed was centrifuged. The centrifugate was checked for complete precipitation by adding Dragendorff’s reagent and the centrifuged mixture was decanted completely. The precipitate was washed with alcohol. The filtrate was discarded and the residue was then treated with 2ml of di-sodium sulphate solution. The brownish-black precipitate formed was then centrifuged. Completion of precipitation was checked by adding 2 drops of disodium sulphate. The residue was dissolved in 2mL of concentrated nitric acid, warming if necessary. This solution was diluted to 10ml with distilled water. Then 1mL of this diluted solution was taken and 5mL of thiourea solution was added. The absorbance was measured at 435nm. The standard curve was made from a stock solution of atropine at 10mg/L with a range from 0 to 1mg/ml. Absorbances were read using a spectrophotometer at 435nm against the white tube prepared under the same conditions by replacing the sample with distilled water. The alkaloid content of the samples was estimated from the linear regression line and expressed in gram equivalents of atropine per 100g of powder.

Quantitative Estimation of flavonoids

      The method described by Patricia et al. [45] was used for the determination of total flavonoids. In a 25-mL flask, 0.75 mL of 5% (w/v) sodium nitrite (NaNO2) was added to 2.5 mL of extract. 0.75 mL of 10% (w/v) aluminium chloride (AlCl3) was added to the mixture and incubated for 6 minutes in the dark. After incubation, 5 mL of sodium hydroxide (1N NaOH) was added and the volume made up to 25 mL. The mixture was shaken vigorously before being assayed using a UV-visible spectrophotometer. The reading was taken at 510 nm. Trials were carried out in triplicate. Flavonoid content was expressed as milligram quercetin equivalent per gram extract (mg Qc-eq/g extract). Quercetin was used here as the reference standard for quantifying total flavonoid content. The total flavonoid content (concentration) was calculated using the formula: £ = 𝐶𝑉𝐷/ 𝑚; £: Content or concentration (mg.AG/g or mg.Qc/g dry extract); C: concentration of the sample given by the spectrophotometer (mg/mL); V: volume of the prepared solution (mL); D: dilution factor; m: mass of the extract (g).

Quantitative Estimation of Phenolic compounds

      The method described by Patricia et al. [47] was used to determine total phenolic compounds. A volume of 2.5 mL of diluted (1/10) Folin-Ciocalteu reagent was added to 30 µL of extract. The mixture was kept for 2 minutes in the dark at room temperature, and then 2 mL of sodium carbonate solution (75 g.L-1) was added. The mixture was then placed for 15 minutes in a water bath at 50°C, and then rapidly cooled. Absorbance was measured at 760 nm, using distilled water as the blank. A calibration line was performed with gallic acid at different concentrations. Each analysis was performed in triplicate and the polyphenol concentration was expressed in milligrams per milliliter of gallic acid equivalent extract (mg/mL). Gallic acid was used here as the reference standard for quantifying total polyphenol content; this quantity was expressed in milligrams of gallic acid equivalent per gram of extracts (mg.eq.GA/g extract). Total polyphenol contents (concentrations) were calculated using the formula: £ = 𝐶𝑉𝐷/ 𝑚; £: Content or concentration (mg.GA/g or mg.Qc/g dry extract); C: concentration of the sample given by the spectrophotometer (mg/mL); V: volume of the prepared solution (mL); D: dilution factor; m: mass of the extract (g)

Quantitative Estimation of tanins

      The tannins are dosed according to the method described by Ba et al.[48]. To 1ml of extract in a test tube are added 5ml of vanillin reagent at 1% (w/v). The tube is left standing for 30 minutes in the dark and the optical density (OD) is read at 415nm against the white. The amount of tannin in the samples is determined using a standard range established from a stock solution of tannic acid (2mg/mL) carried out under the same conditions as the test.

Quantitative Estimation of saponins

      The saponins are dosed according to the method described byMadhu et al. [49]. Test extract was dissolved in 80% methanol, 2ml of Vanilin in ethanol was added, mixed well and the 2ml of 72% sulphuric acid solution was added, mixed well and heated on a water bath at 60 0C for 10min, absorbance was measured at 544nm against reagent blank. Diosgenin is used as a standard material and compared the assay with Diosgenin equivalents.

Quantitative Estimation of Steroids

      The Steroids are dosed according to the method described byMadhu et al. [49].1ml of test extract of steroid solution was transferred into 10 ml volumetric flasks. Sulphuric acid (4N, 2ml) and iron (III) chloride (0.5% w/v, 2 ml), were added, followed by potassium hexacyanoferrate (III) solution (0.5% w/v, 0.5 ml). The mixture was heated in a water-bath maintained at 70±20C for 30 minutes with occasional shaking and diluted to the mark with distilled water. The absorbance was measured at 780 nm against the reagent blank.

Experimental diets

Five experimental diets corresponding to the different treatments were developed using a commercial feed, Skretting, at 50% crude protein. The crude protein ratio of 50% was based on the protein requirements of O. niloticus fry (30-56%) as recommended by Jauncey [50]. Two experimental diets served as controls for our experiments. One was formulated from the original feed to contain 17-alpha methyltestosterone (TMT). The hormone-treated feed was prepared according to the method of Rothbard et al. [51]. Indeed, The hormonal solution was obtained by dissolving 60 mg of hormone (17-alpha methyltestosterone) in 0.7 l of 95% absolute ethanol. The feed was previously reduced to powder and calibrated. Then it was subsequently sprinkled with the hormonal solution at a rate of 60 mg of hormone / kg of feed. The whole was thus mixed to facilitate the incorporation of the hormonal solution into the feed. The mixture was air-dried in the shade for 24 hours to evaporate the alcohol. After drying, the feed was stored in a cold room at 4°C in a plastic bag to preserve the effectiveness of the hormone [52]. However, the second experimental feed called control (T0) which did not receive the hormonal solution was mixed with absolute ethanol and then left to air dry in the shade for 24 hours just like the other feeds. The other 3 were prepared from a commercial feed Skretting, to contain Hibiscus rosa-sinensis leaves ethanolic extract at doses of 100; 110 and 120 mg/kg of feed. The preparation of the different extract-based experimental feeds involved impregnating the feeds with different doses of extracts using the feed-extract mixing technique. After initial homogenization, a volume of 250 ml of 95% ethanol per kg of feed was added to ensure better distribution of the extract in the feed. The experimental feeds were then dried on transparent cloths for 48 h to evaporate the ethanol. This process was carried out away from the sun and at room temperature to preserve its effectiveness [53]. Each test feed was stored in hermetically sealed, labeled boxes.

Experimental procedure

      Seven hundred and fifty (750) O. niloticus fry with an average weight of 2.69 ± 0. 04 g were placed in 15 happas measuring 0.7×0.7×1m placed in a 10x 45 m2 earth pound (Figure 1) at a density of 50 fry per happa and subjected to natural temperature and light conditions. The quantity of feed distributed was set according to the average biomass of fry per week. The fry was fed 30% of their Ichtyo-biomass for the first four weeks of experimentation, 15% the second week, 12% the third week and 10% for the last weeks according to Mareck’s rationing table. The daily ration was divided into 3 meals, from 07:00 to 17:30 with an interval of 5.5 hours [54] and adjusted each week according to the results of weekly population samples. After four weeks of treatment, fry in all treated batches were fed 10% of their biomass with commercial granular feed (Skretting at 30% crude protein). Every morning and evening at 8am and 4pm respectively, the physico-chemical parameters of the water (temperature, pH, and dissolved oxygen) were taken. These parameters, which provide information on water quality, were monitored regularly to ensure optimum rearing conditions for O. niloticus fry.The survival and growth of the fishes sample were monitored from the second week of experimentation, respectively by counting the dead individuals counted and by weighing a sample of 30 individuals taken at random from each of the treatments, at the end of the treatments and then every fortnight until the end of the experiments. The growth performance of O. niloticus fry at the end of this experiment (in terms of Average Weight Gain (AWG), Daily Weight Gain (DWG), Specific Growth Rate (SGR), Total Fish Length (TL), Condition Factor (CF) and Survival Rate (SR) were determined using the following formulae borrowed from various authors [39, 54; 55; 56; 57; 58] These various parameters were calculated at the end of the experiment. These formulae are as follows:

Average Weight Gain: AWG (g) = (Average Final Weight – Average Initial Weight) (g);

Specific Growth Rate (SGR in %.day) = 100. (ln FAW – ln IAW). t-1 with IAW: Initial Average Weight (g); FAW: Final Average Weight (g);

Food Conversion Ratio (FCR): = Rd. (Bf – Bi) -1 with Bi: Initial Biomass (g) Bf: Final Biomass (g) and Rd: Ration or quantity of feed consumed or distributed(g);

Condition Factor (CF) = W×100/ LT3 with W: weight (g), LT: Total length (cm).

Survival Rate (%) = 100x (final number of individuals / initial number of individuals).

Figure 1: Experimental set-up

Sex identification

Thirty (30) fishes from each replicate were randomly selected and prepared for sex identification following the gonad squash technique by Guerrero and Shelton [59]. The experimental fish were dissected, and the gonads were placed on a glass slide. A few drops of acetocarmine were added, and the gonads were gently squashed with a cover slip and then observed under an optical microscope in order to determine the sex based on the gonadal structure (Figure 2).

% male = (number of identified males/total number of fish sample) x 100

Note: The arrows indicate the position of the oocytes in the female gonad.
Figure 2: Gonadal structure of the female (3a) and male (3b) after gonadal squash.
Statistical analysis

      Results are expressed as mean ± standard deviation. The homoscedacity and normality of the datasets were checked beforehand using Hartley’s test. Once the conditions of normality and homoscedacity had been met, a one-way analysis of variance (one-factor ANOVA) was used to analyse the differences between the treatments. The 2-to-2 comparisons were made using the post-test for multiple comparisons of means (Turkey test). Differences were considered significant at p ˂ 0.05. Statistical tests were performed using STATGRAPHICS Centurion version 19.6 software.

Results

Phytochemical characterisation of Hibiscus rosa-sinensis leaves ethanolic extract

Qualitative phytochemical screening revealed the presence of phenolic compounds, flavonoids, alkaloids and tannins, saponins, and steroids in Hibiscus rosa-sinensis leaves ethanolic extract. Quantitative evaluation reveals that Flavonoids with an average 117.27 ± 1.66 mg/g is the most important compound in the extract. The lowest compound is Steroids with an average of 1.88 ±0.05 mg/g (Table 1).

Table 1: Qualitative and quantitative phytochemical composition of Hibiscus rosa-sinensis leaves ethanolicextract

Type of ExtractPhytochemicalsQualitative testQuantitative test (mg/g of extract)
Hibiscus rosa-sinensis leaves ethanolic extractAlcaloids++9.63 ± 0.08
Flavonoids+117.27 ± 1.66
Polyphenols+98.45 ± 1.41
Saponins+2.69±0.07
Steroids+1.88 ±0.05
Tannins++++8.20 ±0.06

Note: [+]: presence of constituent, [++]; moderate concentration of constituent, [+++]; high concentration of constituents, [-] shows absence of constituents.

Physicochemical parameters of water used for culture:

      The main physico-chemical parameters evaluated during this experimental phase were Temperature, Dissolved Oxygen, and pH. It appears that the averages of temperatures and those of dissolved Oxygen and pH remained relatively stable during this phase of experimentation. Indeed, the temperature values oscillated in the average range of 26,45 ± 1.23 °C and 30.29 ± 1.63°C, while the pH values ranged from 7.53 ± 0.55 to 8.39 ±0.22. The average dissolved oxygen values varied from 5.68 ± 0.4 to 5.83 ± 0.34 mg/l. These main values of temperature and dissolved oxygen presented are within the acceptable standards for the breeding of O. niloticus:

Survival and growth characteristics of O. niloticus fry treated with different doses of Hibiscus rosa-sinensis leaves ethanolic extracts

      A comparative analysis at the end of the experimental phase of the different survival rates of O. niloticus fry in control and treated group with different doses of H. rosa-sinensis leaves ethanolic extracts (100; 110 and 120 mg/kg) showed a significant difference (p ˂ 0.05) between treatments (Table 2). Fishes from group treated at 0; 110 and 120 mg/kg of Hibiscus rosa-sinensis leaves ethanolic extracts obtained the highest survival rates (99.62 ± 0.64%; 99.62 ± 0.64%; 99.44 ± 0.96% respectively), while the lowest was obtained from fishes from control group treated with Methyltestosterone at 60 mg/ Kg of feed with an average value of 96.66 ±0.67 %. These results showed that the dose of H. rosa-sinensis leaves ethanolic extract did not significantly affect fishes mortality.

      Growth characteristics varied significantly according to the treatment applied. A comparative analysis of the control group and group treated with different doses of H. rosa-sinensis leaves ethanolic extract (100; 110 and 120 mg/kg) of the different progeny shows a significant difference (p ˂ 0.05) between the treatments (Table 2). The results show that offspring treated with Methyltestosterone at 60 mg/ Kg of feed had a significantly greater effect (p ˂ 0.05) than the other treatments applied in terms of Average Weight Gain (42.54±1.19g), Average Daily Gain (1.01 ±0.02g/d), Specific Growth Rate (6.72±0.45%/d), Average Standard Length (10.43± 1.24cm) and Condition Factor (3.98 ± 0.26(%g/cm3). A comparative analysis of the different group treated with H. rosa-sinensis leaves ethanolic extract (100; 110 and 120 mg/kg) reveals a significant effect of the treatment at the dose of 120 mg/kg on growth performance (Table 2). The poorest growth performance was recorded with the control group (untreated group) and group treated at 110 mg/kg of H. rosa-sinensis leaves ethanolic extract in view of the values obtained for Average Weight Gain (37.62± 1.43g (T0) and 37.68±1.83(HRST 110)) and Average Daily Gain (0.89±0.07g/d (T0) and 0.89 ± 0.09 g/d (HRST 110))  Specific Growth Rate (6.43±0.66 %/d (T0) and 6.44±0.19%/d (HRST 110)), Average Standard Length (10.11± 0.18cm(T0) and 10.12±1.28cm). These results show that the treatment with Methyltestosterone at 60 mg/ Kg of feed gave the best growth performance of the offspring compared with the other treatments applied. However, offspring treated at 120 mg/kg of H. rosa-sinensis leaves ethanolic extract had better growth performanace than those other treated group (100 and 110 mg/kg of H. rosa-sinensis leaves ethanolic extract).

      A comparative analysis of the Consumption Index of the different group fed with feed based on H. rosa-sinensis leaves ethanolic extract compared with the control group showed a significant difference (p˂ 0.05) between the treatments. The offsprings treated with Methyltestosterone at 60 mg/ Kg and those treated at120 mg/kg of H. rosa-sinensis leaves ethanolic extract had a low Consumption Index (1.44±0.05and 1.41±0.45 respectively) compared with the other treated group. The highest value was obtained with the untreated group with an average value of 1.68 ± 0.68.

Table 2: Survival growth parameters and male’s ratio of O. niloticus fry treated with different doses of Hibiscus rosa-sinensis leaves ethanolic extract, compared with untreated group and group treated with Methyltestosterone at 60 mg/ Kg of feed.

Note: Data are expressed as means ± standard deviations. Values with the same superscripts of the same row are not significantly different (p˂0.05). Where, IBW= Initial Body Weight, FBW=Final Body Weight, WG=Weight Gain, ADG=Average Daily Gain, SGR=Specific Growth Rate, FCR=Food Conversion Ratio, SL=Standard Length, CF=Condition Factor, SR=Survival Rate T0= Control treatment 1; TMT= Control treatment 2, Methyltestosterone (60 mg/kg of feed); HRST 100 = H. rosa-sinensis leavesethanolic extract treatment at 100 mg/kg of feed; HRST 110 mg/kg = H. rosa-sinensis leavesethanolic extract treatment at 110 mg/kg of feed; HRST 120 mg/kg = H. rosa-sinensis leaves ethanolic extract treatment at 120 mg/kg of feed.

 Dietary Hibiscus rosa-sinensis leaves ethanolic extract g.kg-1 of diet
ParametersTMT (control)T0(control)HRST 100HRST 110HRST 120P-value
IBW (g)2.69 ± 0. 042.71 ± 0. 022.68 ± 0.082.70 ± 0. 062.68 ± 0. 09–
FBW(g)45.23 ±1.22a40.33±0.57d42.05±2.61c40.38±2.14d43.67±1.50b0.021
AWG (g)42.54±1.19a37.62±1.43d39.37±1.74c37.68±1.83d40.99±1.65b0.017
ADG (g.day1)1.01 ±0.02a0.89±0.07c0.93±0.05b0.89 ±0.09c0.97±0.02ab0.027
SGR (g.day1)6.72±0.45a6.43±0.66c6.55±0.81bc6.44±0.19c6.64±0.27b0.017
FCR1.44±0.05c1.68±0.68a1.51±0.11b1.56±0.73b1.41±0.45c0.021
SL(cm)10.43± 1.24a10.11±0.18c10.23±0.29bc10.12±1.28c10.31±1.46b0,035
CF(%g/cm3)3.98 ± 0.26a3.90 ±0.11b3.92±0.63b3.89±0.17c3.98±0.14a0.046
SR (%)96.66 ±0.67b99.62±0.64a98,50±0.66a99.62±0.64a99.44±0.96a0.037
Male ratio (%)94.44 ± 2.14a47.77±0.25e58.88 ±1.72d68.88±1.44b77.77±1.21c0.041

Sex reversal

      Sexing of fish was done by the standard acetocarmine squash technique of gonads after 60 days of treatment. Microscopic observation after gonadal squash showed that all individuals had normal gonadal development and no intersex individuals were identified. An analysis of the sex ratio obtained following sexing in the different treated group with different doses of H. rosa-sinensis leaves ethanolic extract (100; 110 and 120 mg/kg) compared with the control group (Methyltestosterone-treated group and untreated group) showed a significant effect (p ˂0.05) of treatment on the sex ratio. The highest masculinization rate was obtained in group treated with Methyltestosterone at 60 mg/kg (TMT), with an average rate of 94.44 ± 2.14%. A comparative analysis of the different group treated with H. rosa-sinensis leaves ethanolic extract (100; 110 and 120 mg/kg) reveals a significant effect of the treatment at the dose of 120 mg/kg with an estimated masculinization rate of 77.77 ± 1.21% (Table 3). However, the lowest rate of masculinization was obtained in the untreated batches with an average value of 47.77 ±0.25%.

Table 3: Sex ratio at 60 days post-treatment of O. niloticus offspring treatedwith different doses of H. rosa-sinensis leaves ethanolicextract (100; 110 and 120 mg/kg )compared with the control group (Methyltestosterone-treated group and untreated group)

TreatmentsTotal individualsMaleFemaleMasculinization rate (%)
TMT(control)9085594.44 ± 2.14a
T0 (control)90434747.77 ±0.25e
HRST 100 mg/kg90533758.88 ± 1.72d
HRST 110 mg/kg90622868.88 ±1.44b
HRST 120 mg /kg90702077.77 ± 1.21c
P-value–––0.041

Note:Data are expressed as means ± standard deviations. Values with the same superscripts of the same column are not significantly different (p˂ 0.05).T0= Control treatment 1; TMT= Control treatment 2, Methyltestosterone (60 mg/kg of feed); HRST 100 = H. rosa-sinensis leavesethanolic extract treatment at 100 mg/kg of feed; HRST 110 mg/kg = H. rosa-sinensis leavesethanolic extract treatment at 110 mg/kg of feed; HRST 120 mg/kg = H. rosa-sinensis leaves ethanolic extract treatment at 120 mg/kg of feed.

Discussion

      The main physical and chemical parameters measured during this experiment were temperature, pH and dissolved oxygen. The average values for temperature, pH and dissolved oxygen remained relatively stable during the experimental period. In fact, the temperature values oscillated in the average range of 26.45 ± 1.23 °C and 30.29 ± 1.63°C, while the pH values ranged from7.53 ± 0.55 to 8.39 ±0.22. The average dissolved oxygen values varied from 5.68 ± 0.4 to 5.83 ± 0.34 mg/l. These main temperature and dissolved oxygen values presented are within the acceptable norms for rearing O. niloticus as reported by Omitoyin [60], since the optimum temperature for growth of O. niloticus is between 24 and 28°C, while the pH is between 7- 8. The optimum dissolved oxygen concentration is 5mg/l [61].

      Phytochemical screening revealed the presence of phenolic compounds, flavonoids, alkaloids and tannins, saponins, and steroids in H. rosa-sinensis leaves ethanolic extract. Quantitative evaluation reveals that flavonoids with an average 117.27 ± 1.66 mg/g is the most important compound in the extract. The lowest compound is steroids with an average of 1.88 ± 0.05 mg/g. These results are in line with those obtained by Pekamwar et al. [62], who detected the presence of saponins, tannins, steroids and flavonoids in H. rosa sinensis leaves, flowers and roots extracts. These phytoconstituents might render the androgenic activity of the extracts. Indeed Adhikari et al. [63] revealed that flavonoids, saponins and steroids are natural compounds characterized by androgenic activity. In the same way, Albaho et al. [18] revealed that flavonoids and saponins present in many of plant extracts have been reported to inhibit both the aromatase activity and act as anti-estrogenic compounds. Theses phytochemicals may also compete with endogenous estrogens for binding sites to the estrogen receptor, thereby suppressing estrogen biosynthesis. Consequently, the phytochemicals act as “phytoandrogens” that exert functional effects similar to testosterone in animals, elevating male reproductive characteristics. To this end, the presence of flavonoids and saponins in Hibiscus rosa-sinensis leaves ethanolic extract could be associated with the masculinization rate obtained in group treated with this extract.

      Total survival rates in all treatments and controls were ranging from 99.62± 0.64 to 96.66 ±0.67 % (p ˂ 0.05). Indeed, fishes from group treated at 0; 110 and 120 mg/kg of H. rosa-sinensis leaves ethanolic extracts obtained the highest survival rates (99.62 ± 0.64%; 99.62 ± 0.64%; 99.44 ± 0.96% respectively), while the lowest was obtained from fishes from control group treated with Methyltestosterone at 60 mg/ Kg of feed with an average value of 96.66 ±0.67 %. These high values of the survival rates in the entire group treated with different doses of H. rosa-sinensis ethanolic extract as well as the control group show that these treatments could not have a deleterious effect on the survival of the various offspring. Although a better survival rate was observed in treatments based on H. rosa sinensis ethanolic extract compared with treatment with 17 alpha methyltestosterone. These results are higher to those obtained by Kounde [41] whose work aimed to evaluate the effect of H. rosa sinensis leaves ethanolic extract on the survival and growth characteristics of O. niloticus larvae; with a survival rate values ranging from 81.5% to 95%. This difference could be associated with the different ages of the fishes used and the different doses of extracts applied during the experiments. In fact, the extracts applied had a differential effect on the survival of the offspring. Similarly, sensitivity to a treatment depends on the stage of development. Larvae are more sensitive than juveniles, which has an impact on their survival.

      Growth characteristics varied significantly according to the treatment applied. The results show that offspring treated with Methyltestosterone at 60 mg/ Kg of feed had a significantly greater effect (p ˂ 0.05) than the other treatments applied in terms of Average Weight Gain (42.54±1.19g), Average Daily Gain (1.01 ±0.02g/d), Specific Growth Rate (6.72±0.45%/d), Average Standard Length (10.43± 1.24cm) and Condition Factor (3.98 ± 0.26(%g/cm3). These results show that treatment with 17 alpha methyltestosterone has a greater impact on growth than treatment with Hibiscus rosa sinensis ethanolic extract. This difference is in line with the higher rate of masculinization in group treated with 17 alpha methyltestosterone compared with batches treated with Hibiscus rosa sinensis ethanolic extract. Indeed, O. niloticus males are known to grow faster than females. This could justify this result. However, a comparative analysis of the different group treated with H. rosa-sinensis leaves ethanolic extract (100; 110 and 120 mg/kg) reveals a significant effect of the treatment at the dose of 120 mg/kg on growth performance. These results are higher than those obtained by Jebede [39] in O. niloticus juveniles fed with a diet supplemented with H. rosa sinensis powder, with respective averages of 25.80 g and 0.82 % /day for AWG and SGR. This difference could be associated with the type of treatment applied, which could have a differential effect on the growth performance of the offspring. However, Jebede’s work revealed a significantly greater effect of control group on growth performance compared with group treated with H. rosa sinensis powder.

      An analysis of the sex ratio obtained following sexing in the different treated group with different doses of H. rosa-sinensis leaves ethanolic extract (100; 110 and 120 mg/kg) compared with the control group (17 alpha methyltestosterone-treated group and untreated group) showed a significant effect (p ˂ 0.05) of treatment on the sex ratio. The highest masculinization rate was obtained in group treated with 17 alpha methyltestosterone at 60 mg/kg (TMT), with an average rate of 94.44 ± 2.14%. This result shows that treatment with 17 alpha methyltestosterone is more effective than treatment with H. rosa sinensis ethanolic extracts on O. niloticus fry masculinization. However, a comparative analysis of the different group treated with H. rosa-sinensis leaves ethanolic extract (100; 110 and 120 mg/kg) reveals a significant effect of the treatment at the dose of 120 mg/kg with an estimated masculinization rate of 77.77 ± 1.21%. It should also be noted that the average rate of 47.77 ±0.25% obtained in the untreated group confirms the ratio of 1:1 ratio (i.e. 50% male and 50% female) expected from such a cross. The results show highest percentage of males in all treated groups except the control (untreated group), this infers that H. rosa-sinensis leaves ethanolic extract possess androgenic property which has been found to be effective in O. niloticus. This finding is in agreement with the study done by Phelps and Popma [15] who found higher percentage of male Tilapia when treated with plant extract. This result is higher than those obtained by Kounde [41] in O.niloticus fry treated with vary doses of H. rosa-sinensis ethanolic extract (70; 80 and 100 mg/kg of feed). Indeed, the study of this author revealed a significant effect of the treatment at 100 mg/kg of feed on growth performance and sex ratio (60.82% of males). This difference could be associated with the types of treatment applied (doses of H. rosa-sinensis ethanolic extract) which would have a differential effect on the masculinization of the offspring. The masculine effect might be due to the presence of flavonoids, saponins and steroids in H. rosa-sinensis leaves ethanolic extract, which are natural, compounds characterized by androgenic activity. Albaho et al. [18] revealed that flavonoids and saponins present in many of plant extracts have been reported to inhibit both the aromatase activity and act as anti-estrogenic compounds. Theses phytochemicals may also compete with endogenous estrogens for binding sites to the estrogen receptor, thereby suppressing estrogen biosynthesis. The end result is blocking or attenuating estrogen production, favoring male sex differentiation. Another pathway is that, the bioactive compounds can act as androgen receptor activators. The compounds bind and activate the androgen receptor to function as a transcription factor, which facilitates the expression of the target gene in response to the androgen, subsequently initiating masculinization. This is facilitated by the structural and binding affinity similarities of these phytochemicals and the natural androgen receptor ligands, ie., testosterone and 11-Ketotestostérone. Moreover Olagbende-Dada et al. [64] established the anabolic properties of H. rosa sinensis in immature albino male rats. Indeed, their study results confirmed the androgenic effect of the leaf extracts of H. rosa-sinensis. These androgenic properties can explain the effect of H. rosa-sinensis leaves ethanolic extract on masculinization. However, the highest percentage of males produced by H. rosa-sinensis leaves ethanolic extract was found to be below the ideal requirement of 100% male population. Thus, further studies would be required to establish an ideal treatment regime for production of all-male tilapia population using the H. rosa-sinensis leaves ethanolic extract and to provide conclusive evidence regarding their efficacy to be used as a sex-reversal agent in tilapia culture.

      Before H. rosa sinensis ethanolic extracts can be used as sex-reversal agents by tilapia farmers, others research should be assessed. In view of its androgenic and estrogenic potential, additional studies aimed at inhibiting the constituents with estrogenic activity in order to make the androgenic constituents available are necessary, thus optimising the efficacy of this treatment on the masculinization of O. niloticus juveniles. These studies involve purifying the extract to eliminate undesirable constituents and decrease the content of phytochemicals with known estrogenic activity. Furthermore, research on the efficacy of other extracts as methanol, acetone, or a combination, dosages, and methods of administration should be explored. The commercial application of H. rosa sinensis ethanolic extracts in masculinization of tilapia is also limited by inadequate information regarding the long-term effects of the extracts on the quality of fish’s flesh and other physiological processes (physiological indices as energy metabolism, growth hormones, sex-determination-modulating genes and hormones, and protein turnover). Indeed treatment with H. rosa sinensis ethanolic extracts must be carefully assessed for residues that could affect human health through bioaccumulation in fish tissues as suggested by Gabriel et al. [18].

In addition, it would be interesting to assess the impact of such treatment on the environment directly associated with production. It is known that introducing plant-based additives into aquatic environments could lead to unintended ecological impacts, such as changes in microbial communities or potential bioaccumulation in aquatic organisms [65, 66]. Some plant-derived compounds may possess antimicrobial properties that could disrupt the natural balance of aquatic ecosystems [67].

Conclusion

      The aim of the present study was to evaluate the effect of H. rosa sinensis leaves ethanolic extract on survival, growth and sex reversal of Oreochromis niloticus (Linn. 1758). Phytochemical screening revealed the presence of phenolic compounds, flavonoids, alkaloids, tannins saponins and steroids in H. rosa sinensis leaves ethanolic extract. These phytoconstituents might render the androgenic activity of the extract. The result showed that the dose of extract did not significantly affect offspring mortality. Analysis of the growth parameters revealed that offsprings treated with Methyltestosterone at 60 mg/ Kg feed had a significantly greater effect (p ˂ 0.05) than the other treatments applied in terms of Average Weight Gain (42.54±1.19g), Average Daily Gain (1.01 ±0.02g/d), Specific Growth Rate (6.72±0.45%/d), Average Standard Length (10.43± 1.24cm) and Condition Factor (3.98 ± 0.26(%g/cm3). A comparative analysis of the different group treated with H. rosa-sinensis leaves ethanolic extract (100; 110 and 120 mg/kg ) reveals a significant effect of the treatment at the dose of 120 mg/kg on growth performance. A comparative analysis of the sex ratio of treated group with H. rosa sinensis leaves ethanolic extract revealed a significant effect of the treatment at 120 mg/kg with an estimated masculinization rate of 77.77 ± 1.21%.The highest percentage of males in all treated groups except the control (untreated group) shows thatH. rosa sinensis leaves ethanolic extract enhances the masculinization of O. niloticus fry.Based on our study we conclude that H. rosa sinensis leaves ethanolic extract can be used as an alternative method to produce all-male tilapia population in an environment friendly manner using a natural product. However, the highest percentage of males produced by plant extracts was discovered to be significantly lower than the optimal criterion of a 100% male population. Thus, more research is needed to develop an appropriate treatment regimen for producing all-male tilapia population using plant extracts and to offer solid evidence of their efficiency as a sex-reversal agent in tilapia culture.

Author contribution

      MM conceptualized the study, performed the experiment, analyzed and interpreted data, wrote the first draft, ZP analyzed and interpreted data, FG conceived and designed the experiments, analyzed and interpreted data, NPH conceptualized the study, performed the experiment, TEM Supervision

Declaration of competing interest

      The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper

Acknowledgment statement

      The authors would like to express their gratitude to the promoter of the AQUAKAM production farm, for making the facilities of his farm available to us for this research work. We would also like to thank FADIMATOU GEREHEH, Aquaculture Engineer, for her contribution to this activity.

Funding information

      We confirm that there was no funding for this study. It was self-financed by the authors.

References
  1. Meyer C, (2013) Dictionnaire des Sciences Animales. CIRAD, Montpellier, France. http://dico-sciences-animales.cirad.fr.
  2. El-Sayed AFM (2006) Tilapia culture. Cabi Publishing, London, UK. 294p. http://www.cabi.org/cabdirect/FullTextPDF/2006/20063084667.pdf.
  3. Burel C & Médale F (2014) Quid de l’utilisation des protéines d’origine végétale en aquaculture ?.Oilseeds & fats Crops and Lipids. OCL 2014, 21(4) D406. https://doi.org/10.1051/ocl/2014013.
  4. Trosvik KA, Rawles SD, Thompson KR, Metts LA, Gannam A, Twibell R, and Webster CD (2012) Growth and Body Composition of Nile Tilapia, Oreochromis niloticus, Fry Fed Organic Diets Containing Yeast Extract and Soybean Meal as Replacements for Fish Meal, with and without Supplemental Lysine and Methionine. Journal of the World Aquaculture Society 43: 635–647.  https://doi.org/10.1111/j.1749-7345.2012.00595.x.
  5. FAO (2012). La situation mondiale des pêches et de l’aquaculture. Rome, Italie. http://www.fao.org/docrep/016/i2727f/i2727f00.htm.
  6. El-Sayed AFM (1999) Alternative dietary protein sources for farmed tilapia, Oreochromis spp. Aquaculture 179: 149–168. https://doi.org/10.1016/S0044-8486(99)00159-3.
  7. Lovell T (1995) Opportunities in aquaculture nutrition: Practical considerations in making tilapia feeds. Feed Management 46: 13-22. https://doi.org/10.1007/978-1-4757-1174-5.
  8. Lim C (1989). Practical Feeding – Tilapias. In: Lovell, T. (Ed.), Nutrition and Feeding of Fish. Van Nostrand Reinhold, Inc., New York. p163-183. https://doi.org/10.1007/978-1-4757-1174-5_8.
  9. Devlin R.H., Nagahama Y. (2002). Sex determination and sex differentiation in fish: an overview of genetic, physiological, and environmental influences. Aquaculture. 2002; 208:191-364. https://citeseerx.ist.psu.edu/document?repid=rep1&type=pdf&doi=9968afb47da7511c31717f28b91a142b666da622 .
  10. Nakamura M, Bhandari R K, and Higa M. (2003). The role estrogens play in sex differentiation and sex changes of fish. Fish Physiology and Biochemistry 28: 113–117, 2003. https://www.researchgate.net/profile/Ramji-Bhandari-2/publication/227060155_The_role_estrogens_play_in_sex_determination_and_sex_changes_of_fish/links/55a6866508ae51639c572af0/The-role-estrogens-play-in-sex-determination-and-sex-changes-of-fish.pdf.
  11. Baroiller JF & Jalabert B (1989) Contribution of research in reproductive physiology to the culture of tilapias. Aquatic Living Resources, 2: 105–116. https://www.alr-journal.org/articles/alr/pdf/1989/02/alr89205.pdf.
  12. Mair G C, Abucay JS, Beardmore JA, Skibinski D O F (1995) Growth performance trials of genetically male tilapia (GMT) derived from YY males in Oreochromis niloticus L. on station comparisons with mixed sex and sex reversed male populations. Aquaculture, 137: 313-322. https://doi.org/10.1016/0044-8486(95)01110-2.
  13. Mair G.C. and Little, D.C. (1991). Population control in farmed tilapia. NAGA, ICLARM Quarterly 14(3): 8- 13.
  14. Pandian T.J. and. Sheela, S.G. (1995). Hormonal induction of sex reversal in fish. Aquaculture, 138:1-22. https://doi.org/10.1016/0044-8486(95)01075-0.
  15. Phelps P.R. and Popma, J.T. (2000). Sex reversal of tilapia. In: Costa-pierce AB, Rakocy EK (eds) Tilapia aquaculture in the Americas, 2:39-59.The World Aquaculture Society: Baton Rouge, Louisiana.
  16. Baroiller J. F. & D’Cotta H. (2001). Environment and sex determination in farmed fish. Comparative Biochemestry and Physiology – Part C, 130:399-409. https://doi.org/10.1016/S1532-0456(01)00267-8.
  17. Gabriel NN (2019). Review on the progress in the role of herbal extracts in tilapia culture. Cogent Food Agric 5:1619651. https://doi.org/10.1080/23311932.2019.1619651.
  18. Abaho I., Gabriel N. N., and Izaara A. A (2023). Emerging Sustainable Aquaculture Innovations in Africa. https://doi.org/10.1007/978-981-19-7451-9_7.
  19. Turan F. and Akyurt I. (2005). Effects of androstenedione, a phytoandrogen, on growth and body composition in the african catfish Clarias gariepinus. The Israeli Journal of Aquaculture – Bamidgeh 57(1), 2005, 62-66. https://citeseerx.ist.psu.edu/document?repid=rep1&type=pdf&doi=8334df988a5da3d812a5e88e4c8ca16e643231e3.
  20. Bradbury, R. B., and White, D. E. (1954). Estrogens and related substances in plants. Vitamins Hormones 12:207-233. https://doi.org/10.1016/S0083-6729(08)61013-4.
  21. Rempel, M. A., Schlenk, D. (2008). Effects of environmental estrogens and antiandrogens on endocrine function, gene regulation, and health in fish. International Review of Cell & Molecular Biology, 267, 207–252. https://doi.org/10.1016/S1937-6448(08)00605-9.
  22. Citarasu T. (2010). Herbal biomedicines: A new opportunity for aquaculture industry. Aquaculture International, 18,403–414. https://doi.org/10.1007/s10499-009-9253-7.
  23. Chakraborty, S. B., Hancz, C. (2011). Application of phytochemicals as immunostimulant, antipathogenic and antistress agents in finfish culture. Reviews in Aquaculture, 3, 103–119.   https://doi.org/10.1111/j.1753-5131.2011.01048.x.
  24. Reverter M., Bontemps N., Lecchini D., Banaigs B. & Sasal P.( 2014). Use of plant extracts in fish aquaculture as an alternative to chemotherapy: Current status and future perspectives, Aquaculture, 433, 50–61. https://doi.org/10.1016/j.aquaculture.2014.05.048.
  25. Van Hai  N.( 2015). The use of medicinal plants as immunostimulants in aquaculture: A review, Aquaculture, 446, 88–96. https://doi.org/10.1016/j.aquaculture.2015.03.014.
  26. Jadhav V.M.  , Thorat R.M.,. Kadam V.J. and Sathe N. S.( 2009).Traditional medicinal uses of Hibiscus rosa-sinensis. Journal of Pharmacy Research; 2(8),1220-1222.https://citeseerx.ist.psu.edu/document?repid=rep1&type=pdf&doi=781a11dc61efc3ebf54576c8afd9e18cea681793.
  27. Hirunpanich, V., Utaipat, A., Morales, N. P., Bunyapraphatsara, N., Sato, H., Herunsale, A. (2006).Hypocholesterolemic and antioxidant effects of aqueous extracts from the dried calyx of Hibiscus sabdariffa L. in hypercholesterolemic rats. Journal of Ethnopharmacology.; 103.2: 252–260. https://doi.org/10.1016/j.jep.2005.08.033.
  28. Chang, Y. C., Huang, K. X., Huang, A. C., Ho, Y. C., and Wang, C. J. (2006). Hibiscus anthocyanins-rich extract inhibited LDL oxidation and oxLDL-mediated macrophages apoptosis. Food and Chemical Toxicology. 44.7: 1015–1023. https://doi.org/10.1016/j.fct.2005.12.006
  29. Herrera-Arellano, A., Flores-Romero, S., Chavez-Soto, M. A., and Tortoriello, J. (2004). Effectiveness and tolerability of a standardized extract from Hibiscus sabdariffa in patients with mild to moderate hypertension: a controlled and randomized clinical trial. Phytomedicine; 11.5: 375–382. https://doi.org/10.1016/j.phymed.2004.04.001.
  30. Palaniswamy U. R. (2003). Purslane-Hibiscus.[Online] Available: http://www.lokvani.com. The Asian American Studies Institute, School of Allied Health at the University of Connecticut, Storrs.
  31. Telefor P. B. (1998) Effects of an aqueous extract of Aloe buettneri, Justicia insularis, Hibiscus macranthus, Dicliptera verticillata on some physiological and biochemical parameters of reproduction in immature female rats. J Ethnopharmacol. ; 63:193-200. https://doi.org/10.1016/S0378-8741(98)00062-2.
  32. Missoum A. (2018). An update review on Hibiscus rosa sinensis phytochemistry and medicinal uses. Journal of Ayurvedic and Herbal Medicine. 4(3): 135-146. https://www.ayurvedjournal.com/JAHM_201843_08.pdf.
  33. Jiang, Y. (1998). Effect of petroleum ether extract of Hibiscus rosa-sinensis flowers on early pregnancy and some reproductive hormones in rats. Yunnan Daxue Xuebao, Ziran Kexueban; 20, 162–165. https://europepmc.org/article/cba/313532.
  34. Murthy, D. R. K., Reddy, C. M., & Patil, S. B. (1997). Effect of benzene extract of Hibiscus rosa-sinensis on the oestrous cycle and ovarian activity in albino mice. Biological & Pharmaceutical Bulletin; 20, 756–758. https://doi.org/10.1248/bpb.20.756.
  35. Tan, C. H. (1983). Is Hibiscus rosa sinensis Linn a potential source of anti-fertility agents for males? International Journal of Fertility; 28(4), 247–248. https://europepmc.org/article/med/6142019.
  36. Zhou, M. (1998). Study of anti-fertility agent in petroleum extract of Hibiscus rosa sinensis L. flower. Yunnan Daxue Xuebao. Ziran Kexueban; 20, 170–174.
  37. Batta, S. K.and Shanthakumari, G. (1970).The antifertility effect of Ocimum sanctum and Hibiscus rosa sinensis, Indian J. Med. Res.; 59:, 77-78. PMID: 5097076.
  38. Jana T, Das S, Ray A. (2013). Study of the effects of Hibiscus rosa-sinensis flower extract on the spermatogenesis of male albino rats. J Physiol and Pharmacol Adv.3: 167-71. https://doi.org/10.5455/jppa.20130616115400.
  39. Jegede, T. (2010).Control of reproduction in Oreochromis niloticus (Linnaeus 1758) using Hibiscus rosa-sinensis leaf meal as reproduction inhibitor. Journal of Agricultural Science., 2(4): 149-154. https://www.researchgate.net/profile/Temitope-Jegede/publication/49583889_Control_of_Reproduction_in_Oreochromis_niloticus_Linnaeus_1758_Using_Hibiscus_Rosa-sinensis_Linn_Leaf_Meal_as_Reproduction_Inhibitor/links/552e40dc0cf2acd38cb90ad6/Control-of-Reproduction-in-Oreochromis-niloticus-Linnaeus-1758-Using-Hibiscus-Rosa-sinensis-Linn-Leaf-Meal-as-Reproduction-Inhibitor.pdf?origin=journalDetail&_tp=eyJwYWdlIjoiam91cm5hbERldGFpbCJ9.
  40. Abella, A. T, Espiritu, H. J., Viernes, M., Samelo, V. J., Velasco, R. R., Umagat, R. M., & Gonzales, L. R. (2015). Phytoandrogenic sources for sex inversion of Nile tilapia (Oreochromis niloticus). The Annual International Conference and Exposition of World Aquaculture Society. Asian-Pacific chapter, Jeju, Korea.
  41. Kounde Vanessa. N. (2017).Effets des extraits alcooliques de Hibiscus rosa sinensis sur la survie et le sexe  ratio chez Oreochromis niloticus. Rapport Licence Professionnelle en Production et Santé Animales. Ecole Polytechnique d’Abomey-Calavi (EPAC)-Benin. 51p.
  42. Programme Nationale de Développement Participatif (PNDP) (2015). Plan communal de développement (PCD) de SOA. 197 p. https://www.pndp.org/documents/22_PCD_SOA1.pdf.
  43. Diarra M. N. (2003). Etude phytochimique d’une plante antipaludique utilisée au Mali : Spilanthes oleracea Jacq. (Asteraceae). Thèse de Doctorat, Université de Bamako (Mali) : 78p.
  44. Musa S.A., Buityi J, Mousa M.A.(2000). Immunocytochemical and histological studies on the hypophyseal-gonadal system in the fresh water Nile tilapia Oreochromis niloticus (L.) during sexual maturation and spawning indifferent, habitats. Journal-of-experi-mental-Zoology,Berlin. 2000; 284:3:343-354. https://doi.org/10.1002/(SICI)1097-010X(19990801)284:3<343::AID-JEZ12>3.0.CO;2-V.
  45. Akrout, A., Ayadi, A., & Bonnet, P.(2011). Effect of dietary supplementation with olive oil by-products on growth performance, flesh quality and antioxidant status in Oreochromis niloticus (Nile tilapia). Aquaculture Research, 42(12), 1816-1826.
  46. Sreevidya N. and Mehrotra S. (2003). Spectrophotometric Method for Estimation of Alkaloids Precipitable with Dragendorff’s Reagent in Plant Materials. Journal of AOAC International 2003.Vol. 86, NO. 6.  https://doi.org/10.1093/jaoac/86.6.1124.
  47. Patricia A. K., Barthélemy K. A., Faustin A. K., Brise A. K., Rosmonde A. Y., Herman K. Y., Marcel N. K., Ernest K. A. ( 2020). Comparative physico-chemical study of fermented and unfermented cocoa beans from Côte d’Ivoire. International Journal of Biosciences 16 (1), (2020): 355-362. ISSN: 2222-5234.
  48. Ba K., Tine E., Destain J., Cissé N., Thonart P. (2010). Etude comparative des composés polyphénoliques, du pouvoir antioxydant de différentes variétés de sorgho sénégalais et des enzymes amylolytiques de leur malt. Biotecnology Agronomy Social and envioronmental Sciences 14, 131-139. https://hdl.handle.net/2268/88077
  49. Madhu M., Sailaja V., Satyadev T.N.V.S.S., Satyanarayana M.V.(2016). Quantitative phytochemical analysis of selected medicinal plant species by using various organic solvents. Journal of Pharmacognosy and Phytochemistry 2016; 5(2): 25-29. https://d1wqtxts1xzle7.cloudfront.net/101551120/5-1-31 libre.pdf?
  50. Jauncey K. (2000)..Nutritionnals requirements. M.C.M Beveridge Bj Mc Andrews (eds), Tilapia: Biology and exploitation, Kluwer academic Publishers, 327-375.
  51. Rothbard S., Solnik E., Shabbath S., Amado R., GrabieI. ( 1983) .The technology of mass production of  hormonally sex-inversed all male Tilapias. In Fishelton & Yaron (eds). First International Symposium on Tilapia in Aquaculture. Israel : 425-434.
  52. Varadaraj K., Sindhu Kumari, S., and Pandian, T. J. ( 1994). Comparison of conditions hormonal sex reversaI of Mozambique Tilapias. Progressive Fish-cutlturist 56: 81-90.  https://doi.org/10.1577/1548-8640(1994)056<0081:COCFHS>2.3.CO;2.
  53. Sanches Luís E. F. and Hayashi C. (2001). Effect of feeding frequency on Nile tilapia, Oreochromis niloticus (L.) fries performance during sex reversal in hapas. Maringá, v. 23, n. 4, p. 871876. https://pesquisa.bvsalud.org/portal/resource/pt/vti-725044.
  54. Akinwande, A. A., Dada, A. A.., Moody, F.O. (2011). Effect of dietary administration of the phytochemical “genistein” (3,5,7,3,4 pentahydroxyflavone) on masculine tilapia, Oreochromis niloticus. Elixir Aqua, 33:2231-2233.
  55. Khalil, W. K. B., Hasheesh, W. S., Marie, M-A. S., Abbas, H. H., Zahran, E. A. (2011). Assessment the impact of 17a-methyltestosterone hormone on growth, hormone concentration, molecular and histopathological changes in muscles and testis of Nile tilapia; Oreochromis niloticus. Life Science Journal 8(3), 329-343. https://www.researchgate.net/profile/Hossam-Abbas/publication/216432798_Assessment_the_impact_of_17a-methyltestosterone_hormone_on_growth_hormone_concentration_molecular_and_histopathological_changes_in_muscles_and_testis_of_Nile_tilapiaOreochromis_niloticus/links/0c0919f9953bbf4bb3a87496/Assessment-the-impact-of-17a-methyltestosterone-hormone-on-growth-hormone-concentration-molecular-and-histopathological-changes-in-muscles-and-testis-of-Nile-tilapiaOreochromis-niloticus.pdf.
  56. Kefi, A. S., Kangombe J., Kassam D., Katongo C.(2012). Growth, reproduction and sex ratios in Oreochromis Andersonii (Castelnau, 1861) fed with varying levels of 17aMethyl Testosterone. Journal of Aquaculture Resources Development, 3:8. http://dx.doi.org/10.4172/2155-9546.1000155.
  57. Ogunji J. O., and Wirth M., (2000). Partial Replacement of Fish Meal with Some Alternative Protein Sources in the Diet of Tilapia (Oreochromis niloticus), Session : Technologies for livestock production and Aquaculture. http://ftp3.gwdg.de/pub/tropentag/proceedings/2000/Full%20Papers/Section%20IV/WG%20d/Ogunyi%20J.pdf.
  58. Ahmad M. H., Shalaby A. M. E. Khattab Y. A. E., and Abdel-Tawwab M.(2002). Effects of 17 a–methyltestosterone on growth performance and some physiological changes of Nile tilapia fingerlings (Oreochromis niloticus L.), Egyptian Journal of Aquatic Biology and Fishieries, 4(4) :295-311. https://doi.org/10.21608/ejabf.2002.1736.
  59. Guerrero, R. D., and Shelton W. L. (1974). An aceto-carmine squash method for sexing juvenile fishes. Progressive Fish-Culturist 36:56. https://doi.org/10.1577/1548-8659(1974)36[56:AASMFS]2.0.CO;2.
  60. Omitoyin B.O.(2007). Introduction to fish farming in Nigeria. Ibadan University Press, University of Ibadan, Nigeria, 90 p.
  61. Malcolm C., Beveridje H. & Mcandrew B. J. (2000). Tilapias: biologie and exploitation. Institute of aquaculture. University of stirling, Scotland. Kluwer Academic Publishers. 185 p.
  62. Pekamwar, S. S., Kalyankar, T. M., & Jadhav, A. C. (2013).Hibiscus rosa-sinensis: A review on ornamental plant. World Journal of Pharmacy & Pharmaceutical Sciences; 2, 4719–4727. http://www.wjpps.com/abstract.php?art_id=523&year=2013&issue=VOLUME 2, DECEMBER ISSUE 6.
  63. Adhikari R, Naveen K, Shruthi SD. (2012). A Review on Medicinal Importance of Basella alba L. International Journal of Pharmaceutical Science and Drug Resources. 2012; 4:110-114. http://www.ijpsdr.com/pdf/vol4-issue2/4.pdf.
  64. Olagbende-Dada SO 1,  Ezeobika EN ,  Duru FI.( 2007). Anabolic effect of Hibiscus rosa sinensis Linn. leaf extracts in immature albino male rats. Nigerian Quarterly Journal of Hospital Medicine, 01 Jan 2007, 17(1):5-7.https://doi.org/10.4314/nqjhm.v17i1.12532 
  65. M. Li, D. Wei, S. Huang, et al., “Medicinal Herbs and Phytochemicals to Combat Pathogens in Aquaculture,” Aquaculture International 30, no. 3 (2022): 1239–1259.
  66. J. P. Fernandes, C. M. R. Almeida, M. A. Salgado, M. F. Carvalho, and A. P. Mucha, “Pharmaceutical Compounds in Aquatic Environments—Occurrence, Fate and Bioremediation Prospective,” Toxics 9, no. 10 (2021): 257.
  67. H. Ghafarifarsani, S. H. Hoseinifar, T. Molayemraftar, M. Raeeszadeh, and H. Van Doan, “Pot Marigold (Calendula officinalis) Powder in Rainbow Trout (Oncorhynchus mykiss) Feed: Effects on Growth, Immunity, and, Yersinia ruckeri, Resistance,” Aquaculture Nutrition 2023 (2023): 7785722.
Scroll to Top