Effects of Velvet Beens (Mucuna pruriens) Seeds Methanolic Extract on Survival, Growth Performance and Gonad Development of Nile Tilapia Oreochromis niloticus (Linnaeus, 1758)

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 [3-6]. 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 practices exist and have been developed, including manual sexing, polyculture with predatory species, culture of monosex male populations (by hormonal inversion either by administering androgens via the diet or by balneation, masculinisation via thermal shocks, hybridisation, genetic approaches to producing YY males or YY supermales) [8-10]; sterilisation (through the use of irradiation, chemosterilants and other reproductive inhibitors), intermittent/selective harvesting, the use of slow maturing tilapia species, among others [11]. However, all these population control methods have their shortcomings and limitations. It is therefore necessary to examine a less costly and appropriate technology for solving the problem of uncontrolled tilapia farming using biological inhibiting agents. Early studies have revealed that several medicinal herbs contain phytochemicals (phytoestrogen or phytoandrogens) that are structurally similar to steroid hormones, i.e. 17-ß estradiol (E2) in animals [13]. Recently, these phytochemicals have been reported to induce masculinization, feminization or impair fertility in tilapia species [14-15]. Indeed, herbal extracts could control reproduction in tilapia by impairing fertility through gonads (testes and ovaries) destruction. A study by Jegede and Fagbenro [16] reported swollen spermatids nuclei, increased interstitial cells and focal necrosis in testes; and hydropic degeneration, ruptured follicles, granulomatous inflammation in the interstitium and necrosis ovaries when neem (Azadirachta indica) leaves were incorporated in Tilapia zilli basal diet at 2.0 gkg^-1. Similar findings were reported in O. niloticus fed Carica papaya [17], Hibiscus rosa-sinensis [14], and Aloe vera [18] as well as in O. mossambicus fed dietary Carica papaya and Moringa oleifera respectively [19]. This is an indication that indeed herbal extracts may be used as safe alternative agents to control tilapia early maturity and prolific reproduction in production systems [20].

Mucuna pruriens is a widespread tropical and sub-tropical legume belonging to the order Fabales, family Leguminosae, and subfamily Fabaceae. The plant grows to a height of 3 m to18 m in bushes, hedges, and forests [21-22]. Different parts of this plant have been traditionally used against several diseases worldwide [23]. The herb, Mucuna pruriens (Linnaeus) has various therapeutic uses and aphrodisiac effects in mammals [24]. It has been reported to possess medicinal values and some works indicated the potential use of M. pruriens as alternative source of protein in fish feed [25-27]. The plant has been found to increase libido in men due to its dopaminergic properties [28,29]. The seeds of M. pruriens are considered as astringent, aphrodisiac, nervine and have a high nutritional value [30,31]. There are reports that seed powder of M. pruriens helps in some way against stress, it increases secretion of semen and it acts as a restorative and an invigorating tonic or aphrodisiac in diseases characterized by weakness or loss of sexual power [32]. In another study, it has been reported that M. pruriens seed increased sperm concentration and motility in healthy infertile adults [33]. The seeds of M. pruriens contain tannin, saponin, alkaloid, glycoside, flavonoid, and steroid phytochemicals [34,35]. In particular, the steroids in M. pruriens extracts increased the serum testosterone in animals [36], stimulating androgenic effects, as was observed in rats [37] and fish [35]. The impact of M. pruriens seeds has been explored in various animal species, revealing influences on reproductive indices such as sexual behaviour, gonad growth and gamete quality in rats [24] In deed treatment with ethanolic extracts of M. pruriens seed has resulted a significant and sustained increase in sexual activity, improved mount, intromission and ejaculation, and decreased latencies in normal male rats [32,24]. Mukherjee et al. [35] studied the efficacy of M. pruriens seeds and Asparagus racemosus roots for induction of masculinisation in tilapia. The result suggested that M. pruriens might be regarded to be more potent for induction of masculinization in Nile tilapia as it produced higher percentage of males compared to A. racemosus. However, the action of M. pruriens seeds extracts on gonadal function and spawning of tilapia has very poorly elucidated. Ahmad et al. [38] studied the effect of velvet bean (M. pruriens seeds) extract concentration on individual growth and gonad development of female tilapia fish.  In this study the optimum concentration of velvet bean seed extract 5 ml.kg^-1 of feed resulted in the highest growth in individual length of 16.1 cm and individual weight of 89.43 g, reducing fecundity to 2,206 eggs and egg diameter to 1.11 mm. Considering these aspects, the objective of the present study was to investigate the potential effect of M. pruriens seeds methanolic extract on Survival, growth performance and inhibition of gonad development O. niloticus.

Experimental site 

The study took place at the Laboratory of Aquaculture and Demography of Fisheries Resources of the Institute of Fisheries and Aquatic Sciences of the University of Douala in the locality of Yabassi, capital of the Nkam Department, Littoral Region of Cameroon. It is located between latitudes 9°50' and 10°10' North, and between longitudes 4°20' and 4°40' East, with an average altitude of 15 m to 20 m, corresponding to the NKAM valley. The climate in the Yabassi area is sub-equatorial with tropical tendencies, with two seasons: a dry season from November to June and a rainy season from July to October. The temperature ranges from 24.9°C to 28.2°C, with an average of 27.5°C. 

Collection of plant material 

Six (6) kg of fresh Mucuna pruriens seeds were harvested in their natural habitat (Figure 1) in the city of Yabassi-Cameroon The collected seeds were cleaned and then dried for 21 days away from the sun, as drying in the sun could cause photoreactions that could alter the molecules of certain active components [39]. The seeds were dried under shade and milled into a fine powder. The powder (800 g) was kept in a dry, clean, in glass containers at room temperature until usage. 

 Figure 1 : Mucuna pruriens seeds and powder.

Preparation of Mucuna pruriens seeds methanolic extract

600 g of M. pruriens seeds powder were soaked in 1000 ml methanol at 95% for 72 hours using a mechanical shaker at 250 rpm with constant shaking at intervals as described by [40]. The supernatant was collected and the total volume extracted was filtered using a Watmann filter paper. The filtrate was concentrated by drying it under pressure at a temperature of 45°C for 8 hours using a rotary evaporator. 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 9.67%.

Phytochemical screening

Phytochemical screening was carried out on the basis of characteristic colour tests to identify the main chemical groups. The various phytochemical groups of M. pruriens methanolic extract were characterized using the techniques described by Akrout et al. [41].

Detection of alkaloids (Buchard reaction)

To 1 mL 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 colour indicates the presence of flavonoids.

Detection of polyphenols (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.

Determination of total alkaloids content

The assay was performed using the spectrophotometric method described by Sreevidya & Mehrotra [42]. A quantity of 5 mL of extract solution was taken and the pH was maintained between 2 and 2.5 with dilute HCl. 2 mL 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 2 ml 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 2 mL of concentrated nitric acid, warming if necessary. This solution was diluted to 10 ml with distilled water. Then 1 mL of this diluted solution was taken and 5 mL of thiourea solution was added. The absorbance was measured at 435 nm. The standard curve was made from a stock solution of atropine at 10 mg/L with a range from 0 mg/ml to 1 mg/ml. Absorbances were read using a spectrophotometer at 435 nm 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 100 g of powder.

Determination of total flavonoids content

The method described by Patricia et al. [43] 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).

Determination of total polyphenols

The method described by Patricia et al. [43] was used to determine total polyphenols. 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).

Determination of total tanins

The tannins are dosed according to the method described by Ba et al. [44]. To 1 ml of extract in a test tube are added 5 ml 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 415 nm against the white. The amount of tannin in the samples is determined using a standard range established from a stock solution of tannic acid (2 mg/mL) carried out under the same conditions as the test.

Determination of total saponins

The saponins are dosed according to the method described by Madhu et al. [45]. Test extract were dissolved in 80% methanol, 2 ml of Vanilin in ethanol was added, mixed well and the 2 ml of 72% sulphuric acid solution was added, mixed well and heated on a water bath at 60⁰C for 10 min, absorbance was measured at 544 nm against reagent blank. Diosgenin is used as a standard material and compared the assay with Diosgenin equivalents.

Preparation of experimental feed

Four doses were prepared based on the initial M. prupriens seeds methanolic extract: D1=0.002 mg/kg; D2=0.004 mg/kg; D3=0.006 mg/kg; D4=0.008 mg/kg. Considering the extraction yield of 9.67%, the doses applied were as follows: D1=0.15 mg/kg; D2=0.31 mg/kg; D3=0.48 mg/kg and; D4=0.64. To prepare the dose of D1=0.15 mg/kg, 0.15 mg of crude extract was taken and 100 ml of 75% ethanol was added. As for the other doses, D2=0.31 mg/kg; D3=0.48 mg/kg, 1582 mg and 2110 mg were respectively taken from the crude extract to which  100ml of 75% methanol was added.

Five (05) experimental feeds corresponding to the different treatments were prepared using a base feed: SKRETTING, a commercial floating granulated feed with a diameter of 2 mm and containing 35% protein. The experimental control feed consisted solely of the commercial 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% methanol 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 methanol. This process was carried out away from the sun and at room temperature to preserve its effectiveness [46]. Each test food was stored in hermetically sealed, labeled boxes.

Experimental procedure

375 juveniles of O. niloticus with an average weight of 21.84 ± 1.37 g were placed in 12 happas (cages made with small-mesh nets) measuring 1 x 1 x 1m placed in an earthen pond measuring approximately 75 m², at a density of 25 fishes per happa and subjected to natural temperature and light conditions (Figure 2). The water depth in the pond was maintained at 0.8 m. The quantity of feed distributed was set according to the average biomass of fingerlings per week. The fingerlings were fed 5% of their Ichtyo-biomass for the first four weeks of the experiment, and 4% for the last three weeks, according to Mareck's rationing table. The daily ration was divided into 3 meals, from 07:30 to 17:30 with an interval of 5.5 hours [47] and adjusted every 10 days after the various control fisheries according to changes in the biomass of the subjects. 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. The survival and growth of the subjects were monitored from the second week of experimentation, respectively by counting the dead individuals counted and by weighing a sample of 15 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 juveniles 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),Standard Fish Length (SFL) Condition Factor (K) and Survival Rate (SR) were determined using the following formulae borrowed from various authors [15,47-51]. These different parameters were calculated at the end of the experiment. These formulas are as follows:

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

Specific Growth Rate (SGR) given by SGR (%.d-1) = 100. (lnWf - ln Wi). t^-1 with Wi: Initial average weight (g) Wf : Final average weight (g) ;

Feed 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);

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

Condition factor (K):  = (Wt/ Lt³) *100 (where P = total weight, Lt = total length)

Figure 2: Experimental set-up.

After 45 days post-treatment, a sample of 5 males and 5 females from one of the replicate bathes of each treatment were randomly selected and the remaining individuals from the different batches taken were fed for a further 15 days with the control feed (SKRETTING without containing M. prupriens seeds methanolic extract) in order to observe the post-treatment effect. On the other hand, fishes from the other group treated with M. prupriens seeds methanolic extract respectively at 0.002, 0.004, 0.006 and 0.008 mg/kg of feed were submitted to the various treatments for 15 more days. Males and females were separated using the manual sexing method described by Pelebe [52]. After recording their weights (in order to determine the gonadosomatic index), standard and total lengths, the fish were dissected to determine the maturation of the gonads, fecundity, and egg diameter. After dissection, the gonads (testes and ovaries) were removed, separated by treatment to avoid any confusion and then preserved in 10% formalin solution prior. The gonadosomatic index was determined by the following formula: Gonadosomatic Index (GSI) = (whole organ mass (g)/animal mass (g)) *100. Dissected ovaries were preserved in 10% formalin for 3 weeks, later they were gently agitated to separate the eggs from the ovarian tissues and then the formalin decanted out. The eggs were washed by adding clean water in a beaker containing the eggs, after gentle agitation, the water was filtered out. Entire eggs were put in a clean filter paper and weighed, a sub-sample of the eggs were weighed then counted.  The fecundity of each female fish sampled was determined using the formula: Fecundity = (Total weight of ovary/ Weight of sub sample) X number of mature eggs in sub sample [53]. The diameter of 10 eggs randomly taken from the anterior, middle and posterior parts of the ovary, respectively, was measured using a binocular microscope. The long and short axis of each egg were measured and the mean taken as the diameter of the egg [17].

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 SPSS version 18.0 software.

Phytochemical characterisation of M. pruriens seeds methanolic extract

Qualitative phytochemical screening revealed the presence of phenolic compounds, flavonoids, alkaloids, tannins, saponins and Terpenes in M. prupriens seeds ethanolic extract. Quantitative evaluation of M. prupriens seeds methanolic extracts reveals that tannins with an average of 286.92 ± 2.94 mg/g is the most important compound in the extract of M. prupriens seeds. The lowest compound is Saponins with an average of 0.49 ± 0.04 mg/g (Table 1). 

Table 1: Qualitative and quantitative phytochemical composition of M. pruriens seeds methanolic extract. 

Note: [+]: presence of constituent, [++]; moderate concentration of constituent, [+++]; high concentration of constituents, [-] 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 27.39 ± 0.74°C and 28.74 ± 0.91°C, while the pH values ranged from 7.71 ± 0.1 to 8.28 ± 0.44. The average dissolved oxygen values varied from 5.27 ± 0.5 to 5.63 ± 0.81 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 juveniles treated with different doses of M. pruriens seeds methanolic extracts 

A comparative analysis at the end of the experimental phase of the different survival rates of O. niloticus juveniles in control and treated group with different doses of M. pruriens seeds methanolic extracts (0.002, 0.004, 0.006 and 0.008 mg/Kg) showed a significant difference (p < 0.05) between treatments (Table 2). Fishes from group treated at 0.002 mg/ kg of M. pruriens seeds methanolic extracts obtained the highest survival rates (93.48 ± 4.67 %), while the lowest was obtained from fishes from control group and group treated at 0.008 mg/ kg of M. pruriens seeds methanolic extracts with respectively average value of 82 ± 1.12% and 85.95 ± 4.67%. These results showed that the dose of M. pruriens seeds methanolic 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 M. pruriens seeds methanolic extract (0.002, 0.004, 006 and 0.008 mg/Kg) of the different progeny shows a significant difference (p < 0.05) between the treatments (Table 2). The results show that the offspring treated at 0.006 mg/kg had a significantly greater effect (p < 0.05) than the other treatments applied in terms of Average Weight Gain (24.64 ± 1.07 g), Average Daily Gain (0. 44 ± 0.04 g/d), Specific Growth Rate (1.34 ± 0.04%/d) and Average Fish Length (13.58 ± 1.13 cm). The poorest growth performance was recorded with the control group and group treated at 0.008 mg/Kg of M. pruriens seeds methanolic extract in view of the values obtained for Average Weight Gain (21.83 ± 2.4 g (control), 21.91 ± 2.33 g (group treated at 0.008 mg/kg of MPS), Average Daily Gain (0.39 ± 0.03 g/d (control), 0.39 ± 0.01 g/d (group treated at 0.008 mg/kg of MPS ), Specific Growth Rate (1.23 ± 0.15%/d (control), 1.24 ± 0.33%/d (group treated at 0.008 mg/kg of Mucuna pruriens seeds methanolic extract) . However, fish samples of the control group had obtained a high Condition Factor (with an average of 3.72 ± 0.24) compared with the other treated group. These results show that fish samples treated at 0.006 mg/kg of M. pruriens seeds methanolic extract gave the best growth performance compared with the other treated group with M. pruriens seeds methanolic extract. 

A comparative analysis of the Consumption Index of the different group fed with feed based on M. pruriens seeds methanolic extract compared with the control group showed a significant difference (p < 0.05) between the treatments. The offspring of the untreated group had a low Consumption Index (with an average of 1.43 ± 0.05) compared with the other treated group. The highest value was obtained with the group treated at 0.008 mg/kg of M. pruriens seeds methanolic extract with an average value of 1.71 ± 0.02. 

Table 2: Survival and growth parameters of O. niloticus juveniles treated with different doses of M. pruriens seeds methanolic extract, compared with untreated group.

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, FTL=Fish Total Length, FSL= Fish Standard Length, K =Condition Factor, SR=Survival Rate 

Gonads development inhibition of O. niloticus juveniles fed with experimental diet

An analysis of some reproductive parameters at 45 and 60 days post-treatment of males and females from group treated with different doses of M. pruriens seeds methanolic extract (0.002, 0.004, 006 and 0.008 mg/Kg) compared with control group (untreated group) showed a significant difference (p < 0.05) between treatments (Table 3, Table 4, Table 5 and Table 6). Male and female fishes treated with different doses of M. pruriens seeds methanolic extract showed significantly (p < 0.05) lower gonad/mass weight ratios compared with fishes from control group. This is reflected in the higher gonado somatic index values in the untreated fishes. Mean values of 0.56 ± 0.10% (45 days post-treatment) and 0.59 ± 0.01% (60 days post-treatment) for males and 4.72 ± 0.16 % (45 days post-treatment) and 4.74 ± 0.24 % (60 days post-treatment) for females, respectively. However, analysis of group treated with different doses of M. pruriens seeds methanolic extract revealed significant lower gonado somatic index values in group treated at the dose of 0.006 mg/kg in both males and females with respective mean values of 0.24 ± 0.03 % (45 days post-treatment) and 0.19 ± 0.07% (60 days post-treatment) for males, then 3.02 ± 0.34% (45 days post-treatment) and 2.88 ± 0.18% (60 days post-treatment) for females. Similarly, an analysis of the gonado-somatic index at 60 days post-treatment of satellite males and females from group treated at different doses of M. pruriens seeds methanolic extract compared with control group showed a significant difference (p < 0.05) between treatments (Table 5 and Table 6). Fishes from the control group also obtained higher gonado somatic indices than the treated group. However, analysis of the group treated with different doses of M. pruriens seeds methanolic extract showed significant lower gonado somatic index values in group treated at the dose of 0.006 mg/kg in both males and females, with respective mean values of 0.28 ± 0.05% (for males) and 3.22 ± 0.18% (for females).

Comparative analysis of the weight and size (length) of the testis in fishes samples from treated group and those from control group revealed higher averages for these two parameters in fishes samples from control group, with respective averages of 0.59 ± 0.03 g (weight) and 4.87 ± 0.13 mm (length). The lowest averages were observed in fishes samples from group treated at a dose of 0.006 mg/kg, with respective values of 0.33 ± 0.02 g (weight) and 3.5 ± 0.54 mm (length). Similarly, a comparative analysis of weight and size (length) of the testis of males satellites at 60 days post-treatment showed a significant difference (p < .05) between treatments (Table 4). Males of control group obtained highest averages of this two parameters with respective values of 0.63 ± 0.09 g (weight) and 4.91 ± 0.12 mm (length). The lowest averages were also observed in samples from group treated at a dose of 0.006 mg/kg, with respective values of 0.38 ± 0.07 g (weight) and 3.74 ± 0.31 mm (length). Morphological analysis of the testis showed atrophy in all treated group at different doses of M. pruriens seeds methanolic extract.

Analysis of the fecundity, ovary weight, oocytes diameter, colour and shape also revealed differences between treatments. Samples from the control group had a higher average fecundity (262 ± 2.02 at 45 days post treatment and 284 ± 3.05 at 60 days post treatment), ovary weight mean (1.50 ± 0.10 g at 45 days post treatment and 1.50 ± 0.13 g at 60 days post treatment), and oocystes diameter (2.58 ± 0.12 mm at 45 days post treatment and 2.61 ± 0.26 mm at 60 days post treatment). While those from the samples treated at 0.006 mg/kg of M. pruriens seeds methanolic extract had the lowest average fecundity (187 ± 1.24 at 45 days post treatment and 146 ± 1.32 at 60 days post treatment), ovary weight mean (1.16 ± 0.19 g at 45 days post treatment and 1.13 ± 0.14 g at 60 days post treatment), and oocystes diameter (2.27 ± 0.13 mm at 45 days post treatment and 2.21 ± 0.16 mm at 60 days post treatment). Observations on oocytes colour revealed a difference between treatments.  Fishes samples from the control group and those from the group treated at 0.002 mg/kg and 0.004 mg/kg of M. pruriens seeds methanolic extract respectively showed oocytes with a yellowish colour at 45 and 60 days post treatment. On the other hand, samples from group treated at 0.006 mg/kg and 0.008 mg/kg of M. pruriens seeds methanolic extract respectively showed whitish-coloured oocytes during the same period, indicating that the treatment dose had an impact on oocyte quality. However, observations on oocytes morphology revealed that oocystes had an oval shape in all the treatment at 45 and 60 days post treatment. Morphological analysis of the gonads showed atrophy in all treated group at different doses of M. pruriens seeds methanolic extract. Observation of female gonadal morphology revealed a difference between treatments at 45 and 60 days post treatment (Table 5). Fishes from the control groups showed two well-developed oocyte envelopes. However, gonad atrophy was observed in all treated group at different doses of M. pruriens seeds methanolic extract. 

Table 3: Gonado somatic index and characteristics description of testis of O. niloticus males of different treatment group at 45 and 60 days post treatment.

Data are expressed as means ± standard deviations. Values of the same column with different superscripts differ significantly (P < 0.05) 

Table 4: Gonado somatic index and characteristics description of testis of O. niloticus males of different treatment group after 45 days post isolation (+15 days of feeding at 0%).

Data are expressed as means ± standard deviations. Values of the same column with different superscripts differ significantly (P < 0.05) 

Table 5: Reproductive parameter and ovary observations of Oreochromis niloticus females of different treatment group at 45 and 60 days post treatment.

Data are expressed as means ± standard deviations. Values of the same column with different superscripts differ significantly (P < 0.05) 

Table 6: Reproductive parameter and ovary observations of Oreochromis niloticus females of different treatment group after 45 days post isolation (+15 days of feeding at 0%).

Data are expressed as means ± standard deviations. Values of the same column with different superscripts differ significantly (P < 0.05)

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 27.39 ± 0.74°C and 28.74 ± 0.91°C, while the pH values ranged from 7.71 ± 0.1 to 8.28 ± 0.44. The average dissolved oxygen values varied from 5.27 ± 0.5 to 5.63 ± 0.81 mg/l. These main temperature and dissolved oxygen values presented are within the acceptable norms for rearing O. niloticus as reported by Omitoyin [54], 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 5 mg/l [55].

Phytochemical screening revealed the presence of phenolic compounds, flavonoids, alkaloids, tannins, saponins, and terpenes in M. prupriens seeds methanolic extract. Quantitative evaluation of M. prupriens seeds methanolic extracts reveals that tannins with an average of 286.92 ± 2.94 mg/g is the most important compound in the extract of M. prupriens seeds. The lowest compound is Saponins with an average of 0.49 ± 0.04 mg/g. These results are in line with those obtained by Mukherjee et al. [35], who detected the presence of flavonoids, saponins, tannins, and flavonoids in M. prupriens seeds ethanolic extracts. These phytoconstituents might render the antifertility activity of the extracts. Previous phytochemical investigation revealed an array of alkaloids and flavonoids in the seed such as the alkaloids bufotenin, dimethyltryptamine, tetrahydroquinolone alkaloids, stizolamine, mucunine, mucunadine, prurienidine, nicotine and the flavonoids namely genistein, medicarpin, kievitone, cajanol etc [56-57].

Total survival rates in all treatments and control were ranging from 93.48 ± 4.67% to 84.55 ± 4.77% (p < 0.05). Indeed fishes from group treated at 0.002 mg/ kg of M. pruriens seeds methanolic extracts obtained the highest survival rates (93.48 ± 4.67%), while the lowest was obtained from fishes from control group and group treated at 0.008 mg/ kg of M. pruriens seeds methanolic extracts with respectively average value of 84.55 ± 4.77% and 85.95 ± 4.67%. Those high values of the survival rates in the entire group treated with different doses of M. prupriens seeds methanolic extract as well as the control group show that treatments could not have a deleterious effect on the survival of the various offspring. In another study as well, toxic changes, stress and changes in behavior were not observed in rats treated with differential doses of ethanolic extracts of M. pruriens [32]. In a feeding trial with Nile tilapia, fish fed diets containing differentially processed Mucuna seeds for 56 days, showed no mortality for the entire period of experiment [63]. However the survival rate obtained in this study are lower than those obtained by Mukherjee et al. [34] in Nile tilapia juvenile subjected to dietary treatment with powdered M. pruriens seeds (0.0, 2.0, 3.5 and 5.0 g/kg feed) and  immersion treatment with aqueous extract of the plant seeds (0.02, 0.035 and 0.05 g/l). This difference could be associated with the types of treatment applied, which would have a differential effect on survival rate of the offspring.

Growth characteristics varied significantly according to the treatment applied. The results show that fish sample treated at 0.006 mg/kg of M. prupriens seeds methanolic extract had a significantly greater effect (p < 0.05) than the other treatments applied in terms of Average Weight Gain (24.64 ± 1.07 g), Average Daily Gain (0. 44 ± 0.04 g/d), Specific Growth Rate (1.34 ± 0.04%/d) and Average Fish Length (13.58 ± 1.13 cm). These result is higher than those obtained by Saiyad et al. [58] in O. mozambicus juveniles fed with M. prupriens seed meal enriched diet at 2, 4, 6 and 8 g/kg feed. This difference could be associated with the types of treatment applied, the species, the age of the fishes used which would have a differential effect on the growth performance of the offspring. Recently Ojha et al. [59], has been reported that the M. pruriens seed meal significantly enhances growth performance, metabolic activity, and immune response in Labeo rohita. At the same time Siddhuraju et al. [60], stated that the higher inclusion rate of M. pruriens seed meal significantly reduced the growth parameters in Cyprinus carpio because most of the plant-based feed stuffs have a wide variety of anti-nutritional factors such as phytin, non-starch polysaccharides (NSP) and protease inhibitors, which may impair nutrient utilization, as well as impair fish growth performance and health [61].

An analysis of some reproductive performance at 45 and 60 days post-treatment of males and females from group treated at different doses of M. pruriens seeds methanolic extract compared with control group (untreated group) showed a significant difference (p < 0.05) between treatments. Indeed, reproductive performance of male (gonadosomatic index (GSI), weight and size (length) of the testis) and females (gonadosomatic index (GSI), fecundity, mean egg diameter, female ovary weight) O. niloticus were significantly (p < 0.05) decreased in those treated group when compared with the control at 45 and 60 days post-treatment. However, analysis of fish samples treated with M. pruriens seeds methanolic extract revealed a significantly higher effect of the 0.006 mg/kg dose on reproductive performance inhibition of male and females.

Gonad weight and gonadosomatic index (GSI) are indicators of gonadal maturation in fishes. Results showed that males and females GSI values decreased (p < 0.05) as the dose of M. pruriens seeds methanolic extract increased which was similarly reported by Jegede and Fagbenro [16] and is attributable to the poor development of testes tissues and ovarian tissues as suggested by Cumaranatunga and Thabrew [62]. On the other hand, a significant decrease occurred in GSI of females agreed with the finding of Jegede and Fagbenro [16] and Temitope [63] who reported a significant decrease in GSI of Nile tilapia females treated with other medicinal plants such as neem (Azadirachta indica) and Hibiscus (Hibiscus rosa Sinensis) leaf.

Analysis of fecundity and egg diameter showed a significant difference between treatments (p < 0.05). Fishes samples treated at 0.006 mg/kg with M. pruriens seeds methanolic extract had the lowest mean fecundity of 187 ± 1.24 (45 days post-treatment) and 146 ± 1.32 (60 days post-treatment) with egg diameter of 1.27 ± 0.13 (45 days post-treatment ) and 1.21 ± 0.16 (60 days post-treatment) egg diameter. While, fishes samples of the control group showed highest mean fecundity of 262 ± 2.02 (45 days post-treatment) and 284 ± 3.05 (60 days post-treatment) with egg diameter of 2.58 ± 0.12 (45 days post-treatment) and 2.61 ± 0.26 (60 days post-treatment). These results in fishes samples treated at 0.006 mg/kg with M. pruriens seeds methanolic extract, although lower than those obtained by Madan Prasad [64] on O. niloticus juveniles fed diets supplemented with different doses of Aloe vera latex (i.e. respective averages of 258.0 ± 2.12 (fecundity) and 2.33 ± 1.77 mm (oocyte size), are in line with his hypothesis that as the dose increases, fecundity decreases. This result obtained in control group is in agreement with Coward and Bromage [65] who reported that eggs produced by incubating females of O. niloticus normally exceed 2 mm in diameter and that fecundity is generally less than 350 in these females. Results obtained on the fecundity and egg diameter of the fish corroborate results obtained on the total length, ovary weight and gonadosomatic index. This is in view of the fact that fecundity in fish has been reported to have positive significant correlation with total length and ovary weight [66]. This implies that a decrease in the ovary weight and egg diameter will result in decrease in fecundity. This assertion is supported by Phukon and Biswas [67] and Buragohain and Goswami [68].

Analysis of the morphological characteristics of the male and female gonads revealed atrophy of the gonads in the entire group treated with M. pruriens seeds methanolic extract. These observations are similar to those of Madan Prasad [64] on O. niloticus juveniles fed diets supplemented with different doses of Aloe vera latex. The gonad atrophies observed in the treated group reflect the impact of the various treatments on gonad inhibition. In addition to the atrophy, other morphological differences were observed in the females. Samples from the control group showed two well-developed oocyte envelopes. However, fishes treated with M. pruriens seeds methanolic extract showed malformation of the second oocyte envelope. However, morphological observations of eggs from the various treatments did not reveal any deleterious changes in egg shape. In fact, a normal oval egg shape was observed. This observation corroborates the study carried out by Arrignon [69] on Tilapia. Analysis of oocyte coloration showed a difference between treatments. Fishes samples from control group as well as those treated at doses of 0.002, 0.004 mg/kg showed a yellowish coloration. On the other hand, oocytes from group treated with 0.006 mg/kg and 0.008 mg/kg respectively were whitish in color. These observations show that the normal olive green colour of O. niloticus eggs was not maintained as stipulated by Madan Prasad [64]. This difference in colour reveals an impact of the treatment dose on the quality of the oocytes.

Figure 3: Gonads morphological traits Males (B) and females (A) O. niloticus treated at different doses of M. pruriens seeds methanolic extract.

T0: untreated group; T1: group treated with M. pruriens seeds methanolic extract at 0.002 mg/kg; T2= group treated with M. pruriens seeds methanolic extract at 0.004 mg/kg; T3= group treated with M. pruriens seeds methanolic extract at 0.006 mg/kg; T4= group treated with M. pruriens seeds methanolic extract at 0.008 mg/kg

The aim of the present study was to evaluate the effect of M. pruriens seeds methanolic extract on the survival, growth performance and inhibition of gonad development of O. niloticus juveniles. Phytochemical screening revealed the presence of phenolic compounds, flavonoids, alkaloids, tannins, saponins, and Terpenes in M. prupriens seeds methanolic extract. These phytoconstituents might render the antifertility activity of the extract. This work showed that the dose of extract did not significantly affect juvenile mortality. Growth characteristics analysis revealed that fish sample treated at 0.006 mg/kg of M. prupriens seeds methanolic extract had a significantly greater effect (p < 0.05) than the other treatments applied. However, reproductive parameters of male (gonadosomatic index (GSI), weight and size (length) of the testis) and females (gonadosomatic index (GSI), fecundity, mean egg diameter, female ovary weight) O. niloticus were significantly (p < 0.05) decreased in treated group when compared with the control at 45 and 60 days post-treatment. Analysis of the morphological characteristics of the male and female gonads revealed atrophy of the gonads in the entire group treated with M. pruriens seeds methanolic extract. The gonad atrophies observed in the treated group reflect the impact of the various treatments on gonad inhibition Based on our study we conclude that M. pruriens seeds can be used as a natural agent to control the reproduction of Nile tilapia and overcome the problem of early maturation.

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

The authors would like to express their gratitude to the field technicians of the Laboratory of Aquaculture and Demography of Fisheries Resources of the Institute of Fisheries and Aquatic Sciences of the University of Douala for their assistance during the research.

None.

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