Concentration and time of feeding with 17- -methyltestosterone oil diluted and incorporated to the feed for masculinization of Nile tilapia

. Tilapia masculinization can be induced by oral administration of  - methyltestosterone (MT), which is commonly dissolved in ethanol to be added to the feed. However, there are many benefits in using alternative vehicles, such as oil. The incorporation time, vehicles quantity, safety for handlers, fish and the environment are favorable factors. In fry fed for 35 days under temperature control, we found that masculinization rate was similar in both incorporation vehicles of MT (oil or ethanol) in the concentrations studied (30 and 60 mg MT kg -1 feed). In an experiment, using hormone oil dissolution and oral administration at 30 mg MT kg -1 feed, it was observed that the longer the administration time, the lower the coefficient of variation in the masculinization rate. Therefore, administration for 32 days showed the lowest variability in the masculinization rate (99.8 ± 0.5 %), compared to 24 (98.5 ± 3.0 %), 16 (97.0 ± 6.0 %) and 8 (89.0 ± 8.8 %) days. The field experiment confirmed the results obtained in the lab. We concluded that the oil can be used as MT vehicle and we recommend to dispense it at the lowest hormonal concentration (30 mg MT kg -1 feed) for 32 days for tilapia


Introduction
Nile tilapia (Oreochromis niloticus) is regarded as one of the most important aquaculture species that must be studied for the improvement of the several available production systems, since it is within the capacity of the consumers around the globe in terms of nutritional value and prices (Amer, El-Nabawy, Gouda, & Dawood, 2021;Food and Agriculture Organization [FAO], 2018).The presence of female tilapia in the production systems is undesired, since it can lead to overpopulations of unevenly sized fish in the production units, due to the deviation of growth energy for gonadal development and reproduction, thus tilapia females do not grow as valuable as males (Pandian & Sheela, 1995;Homklin, Ong, & Limpiyakorn, 2012;El-Greisy & El-Gamal, 2012;El-Sayed, 2006).In addition, its rapid proliferation harms the final production because of competition for food and oxygen, causing heterogeneity of the population (Popma & Green, 1990).Therefore, higher yields and more uniformly-sized fish are obtained in production by the use of all-male tilapia.
The masculinization protocol relies on several factors including route of administration, duration, and time of treatment, concentration, and general characteristics of the commercial hormones (Piferrer, 2001).In tilapia masculinization with oral administration of MT, the recommendations range from 30 to 60 mg MT kg feed, in periods of 30 to 60 days (Celik, Guner, & Celik, 2011;Clemens & Inslee, 1968;Jensi, Marx, Rajkumar, Shakila, & Chidambaram, 2016;Mainardes-Pinto, Verani, Campos, & Silva, 2000;Rima, Rahman, & Sarker, 2017;Singh, Saini, & Sharma, 2018;Yamazaki, 1983), in four or more daily feedings (Meurer, Bombardelli, Paixão, Silva, & Santos, 2012).The field recommendation by the scientific community and practiced by most of tilapia hatcheries is that the hormone is first diluted in ethanol and then incorporated into the feed by mixing manually or in proper mixing equipment such as mixers.Following, the feed is exposed to air for ethanol evaporation (Guerrero III, 1975).
A hormonal product was recently launched on the market for incorporation in tilapia feed (in the sexual reversion phase) in oil (30 mL per kilo of feed), at 30 mg MT kg -1 feed.The commercial product seems to have advantages in the sense of not requiring the handling of large amounts of ethanol (half a liter per kilo of feed).In addition, the use of oil eliminates the risk of the presence of ethanol in the feed, which can cause liver damage, reducing health and survival of fingerlings (Valentim-Zabott et al., 2008).However, this product is diluted by 50% with lactose in order to help its incorporation in feed.To better understand the effect of dissolving pure MT with oil, we analyzed the masculinization rate and performance of Nile tilapia with both vehicle of MT incorporation (oil and ethanol), in different concentrations and time of administration under controlled temperature (laboratory conditions).

Trial 1: Solvents and concentrations experiment
The experiment was conducted at the Aquaculture Laboratory of Embrapa Western Agriculture, Dourados, MS.Nile tilapia larvae, GIFT strain, came from a commercial hatchery.The larvae (average weight 0.012 g) were distributed in twenty fiber tanks of 300-L each (400 fish per tank).The water flow was constant at 0.8 L min. - , and an individual stone diffuser per tank was used to ensure dissolved oxygen saturation above 90%.
The experimental design was completely randomized, with five treatments and four replicates.The treatments were two concentrations of synthetic hormone 17--methyltestosterone (30 and 60 mg kg -1 ), two hormone incorporation vehicles (commercial ethanol 96 GL and soybean oil), and the control (hormone-free diet).
The 17-alpha-methyltestosterone (MT) was weighed at the test concentrations (60.0 or 30.0 mg) and diluted in oil (16 mL of oil per kg of feed) or ethanol (0.44 L of ethanol per kg of feed).The mixture of the diluted hormone in one kilo of the commercial powdered feed (55% Crude protein, CP) was processed manually until obtained lumps-free feed.The experimental feed containing the hormone diluted in oil was ready for use, while the feed diluted with ethanol was distributed in metallic trays lined with brown paper, forming a thin layer of feed for efficient drying.The trays were placed in an oven with forced air circulation at 50°C for 24 hours for ethanol evaporation.All feeds were kept under refrigeration (4 o C) and protected from light until used.The proximate composition of experimental diets was estimated following standard protocols (Association of Official Agricultural Chemists International [AOAC], 2012).Dietary gross energy was analyzed using a bomb calorimeter (IKA-C 6000 Isoperibol).The proximate composition of all experimental diets is shown in Table 1.Fish were hand-fed eight times a day (8:00 am, 9:00 am, 10:00 am, 11:00 am, 1:00 pm, 2:00 pm, 3:00 pm and 4:00 pm) for 28 days.The daily feeding rate (15% of biomass) was adjusted once a week, when 10% of fish from a random replicate of each treatment were weighed.After completion of hormonal treatment, fish were individually counted and weighted per tank.The animals (average weight) remained in the same tanks, receiving pelleted feed (0.8 -1.5 mm; 36% CP) until apparent satiety, twice a day (8:00 am and 4:00 pm).After a 35-day growth period, 100 fish in each replicate were anesthetized with eugenol (1:50,000 v/v) and euthanized by spinal cord transection (Owatari et al., 2022) to evaluate the efficiency of the treatments on masculinization.Thus, sex ratios were determined by microscopic analysis (100 magnification) of gonadal squash.Ovaries contain oocytes characterized by their exocytosis, previtellogenic or vitellogenic features.Male gonads were clearly distinguished due to their lobular configuration (Kubitza, 2000;Nakamura & Nagahama, 1989;Phelps & Popma, 2000).
Trial 2: Period of 17--methyltestosterone administration (30 mg MT kg -1 feed) diluted with oil The study was conducted for 66 days.Nile tilapia larvae (initial average weight 0.014 g) were distributed in a completely randomized design, consisting of five treatments with four replicates and 500 fish per replicate.Twenty circular fiber boxes (300-L) were used in a system with temperature control and oxygen supply.The treatments comprised five periods of MT administration (0, 8, 16, 24 and 32 days).
The hormone (30 mg MT kg -1 ) was diluted with oil and incorporated into the feed following the same procedure as in the previous experiment.The fish were fed eight times a day (8:00 am, 9:00 am, 10:00 am, 11:00 am, 1:00 pm, 2:00 pm, 3:00, and 4:00 pm).The daily feeding rate was adjusted according to the growth of the animals (20 and 15% of biomass, in the first and other weeks, respectively), in the same way as Trial 1.

Trial 3: field validation
Larvae of O. niloticus (n = 8,000) were transferred five days after hatching to two hapas (2 m 2 ) set in earthen pond (1,000 m 2 ) in March-April, 2021.The pond was equipped with an aerator and there was continuous water inlet (8% renewal rate/ day).The average water temperature and oxygen dissolved were 26.5 ± 0.5°C and 6.63 ± 0.4 mg L -1 , respectively.Feeds containing MT were prepared using the hormone (30 mg MT kg -1 ).In this process, for the preparation of 25 Kg of feed, MT was diluted in oil (16 mL of oil per kg of feed) and the mixture was incorporated into eight kilos of feed by hand.This feed was added to the 17 kg remaining, using a sieve for a homogeneous incorporation.Finally, it was mixed mechanically for five minutes in an adapted concrete mixer.After this procedure, it was ready for use.
Fish were fed at 20-15% body weight daily, seven days per week, for the entire treatment period.A daily feed was divided into eight portions given at 08:00 am, 9:00 am, 10:00 am, 11:00 am, 1:00 pm, 2:00, 3:00, and 4:00 pm.Fish (approximately 400 fish per period) were collected on days 8, 16, 24 and 32 after starting hormonal treatment.Collected fish were stocked in other four hapas (2 m2) for growth, being separated by treatment.All hapas were placed in a pond (3,000 m2) which was not used for masculinization.Fish from each hapa were harvested once reached about 10 g.It was used the same methodology for the analysis of sex (n=100/treatment) as used in other trials.

Statistical analysis
The effects of hormone treatment on the weight gain, survival rate, feed conversion ratio, and sex ratios were tested for normality using a Shapiro-Wilk's test and fulfilling the assumptions for normality; data were analyzed by variance analysis (ANOVA).Differences among treatments were identified using Tukey test (p=0.05).All statistical analyses were performed in RStudio software (R Core Team, 2021).

Results and discussion
In testing solvents and concentrations of MT administration, feed conversion ratio, and survival rate of the different treatments were not significantly different from each other (Table 2).The control group (hormone-free diet) had the lowest masculinization rate (51.5%), while the masculinization rate did not differ between hormone concentrations and solvents used (Table 2).The treatments affected the weight gain; the lowest weight gain was obtained with 60 MT kg -1 feed diluted with oil (Table 2).However, no clear relationship was found between the concentration and the solvent that justified the obtained results.Therefore, the differences found may have other sources of variation, in addition to the treatments.The results indicated ethanol can be replaced by oil to dissolve 17--methyltestosterone in the both tested concentrations (60 and 30 mg MT kg -1 feed), for Nile tilapia fry (Oreochromis niloticus) masculinization.In Brazil, most of commercial tilapia hatcheries and juvenile farmers commonly use 60 mg MT kg -1 feed diluted in ethanol.The use of different solvents can influence the masculinization rate (Varadaj & Pangian, 1991), but this influence was not verified in the present study.This fact probably occurred due to the quality of the MT incorporation process, in which the oil was uniformly adsorbed in the feed and absorbed by the fish, without showing apparent losses of oil and, consequently, of the hormone in the water.Therefore, we presume that the concentration of 30 mg MT kg -1 feed was enough to induce a masculinization rate equal to ethanol, and above 98%.
Weight gain and survival rate have no difference among the periods of MT administration (Table 3).Based on the results, 30 mg MT kg -1 feed administrated for 32 days promoted better feed conversion ratio compared to control group.Frequencies of male in 8, 16, 24 and 32 days were significantly higher than the control group (Table 3).It was observed that the MT administration (30 mg MT kg -1 feed) for a period of 32 days showed greater accuracy of masculinization, as it presented a lower coefficient of variation (Figure 1).Table 3. Weight gain (WG), feed conversion ratio (FCR), survival rate (SR), and male frequency (MF) of tilapia at different times of 17α-methyltestosterone administration (30 mg MT kg -1 feed) after a 32-day feeding period.In general, a concentration of 60 mg kg -1 of feed is used for 28 days in Nile tilapia fry, treated with MT administered orally and feed added in ethanol (Beardmore, Mair, & Lewis, 2001;Bombardelli, Hayashi, & Meurer, 2004;Celik, Guner, & Celik, 2011;Ferdous & Ali, 2011;Popma & Green, 1990).However, lower concentration administration (14 mg MT kg -1 feed) was recommended by Phelps and Okoko (2011) to obtain Acta Scientiarum.Animal Sciences, v. 46, e62366, 2024  95% males after 28 days of feeding.In addition, no difference was observed between 30 and 60 mg MT kg -1 feed, on the frequency of Nile tilapia males treated for 45 days (Mainardes-Pinto et al., 2000).In Tilapia mossambica, according to Nakamura (1975), methyltestosterone at 50 mg kg -1 feed, given for 19 days, is capable of inducing a complete masculinization.In the present study, the administration periods of 16, 24, and 32 days induced a masculinization rate above 95% using MT diluted with oil at 30 mg MT kg -1 feed, and it was observed that the longer the administration time, the lower the coefficient of variation in the masculinization rate.Tilapia nilotica fed with MT (30 mg kg -1 feed) for 59 days showed 100% sex reversal (Tayamen & Shelton, 1978).According Vinarukwong, Lukkana, and Wongtavatchai (2018), in Nile tilapia, the percent of phenotypic males increased with the longer duration of hormone treatment.Considering the higher variability in the masculinization rate could lead to a greater population of fish with uneven sizes in production units, we recommend a 32-days treatment period using MT diluted with oil at 30 mg MT kg -1 feed.This is in accordance with Macintosh, Varghese, and Rao (1985), who stated that although sex reversion of 0reochromis mossambicus can occur using only 21 or 28-day treatment periods and a daily rate of 3% body weight, it is clearly advantageous to use a higher feeding rate and/or to extend the length of treatment to more than 30 days.
Survival rate was not affected by concentrations, vehicles, and time of MT administration.These results are in line with the previous study, which demonstrated MT administration did not reflect negatively on tilapia survival rate when compared to group that did not receive any MT (Ahmad et al., 2002;Mainardes-Pinto et al., 2000;McGeachin, Robinson, & Neill, 1987;Soltan et al., 2013;Rima, Rahman, & Sarker, 2017).The low survival rate observed in the present study may be due to the individual fish characteristics (Celik, Guner, & Celik, 2011), and to the establishment of hierarchies in feeding among the fish (Vera Cruz & Mair, 1994).
Regarding the composition of the diet, the addition of oil increased the ether extract and, on the contrary, the use of ethanol decreased the ether extract from the diet.This is because ethanol is a solvent and, depending on the MT incorporation procedure, can extract lipids from the diet.Although the variations obtained in the ether extract were not enough to influence the gross energy, lipids are a source of essential fatty acids, important for the healthy development of fish (Qiang et al., 2017).Therefore, the possibility of changes of essential nutrients from the original formulations must be considered and evaluated in further studies.
Under field conditions, masculinization rate in fish was over 90% when administrated for 16, 24, and 32 days (30 mg MT kg -1 feed) (Figure 2).Based on the results of laboratory experiments and field validation, it is possible to dissolve MT (100% pure) with pure oil to produce monosex Nile tilapia.Since masculinization rate and tilapia performance at 30 mg MT kg -1 feed showed no difference to 60 mg MT kg -1 feed, the lowest concentration is considered the ideal, in order to reduce risks to workers, water and the environment.In the field, the evaporation of ethanol is commonly carried out at room temperature, taking around two to three days.In this sense, the standard process of incorporating MT with oil (with criteria to guarantee the homogeneous mixture of the defined amount of MT in the feed) also brings benefits with regard to the reduction of time in the incorporation of the diet with MT, in addition to using less vehicle.Reducing the amount of hormone and vehicle, preparation time, and labor, results in lower masculinization costs, adding one more advantage to the use of oil.Under favorable temperature conditions for tilapia, the 32 days of MT feeding period is recommended to achieve better feed conversion of Nile tilapia and greater precision in the masculinization rate.This information may favor many farmers of male tilapia juveniles.

Conclusion
Feeding Nile tilapia for 32 days with a diet containing soybean oil (16 ml oil kg -1 feed) as a vehicle for the hormone 17--methyltestosterone (30 mg MT kg -1 feed) proved to be efficient in the process of sex reversal.

Table 1 .
Treatments and analyzed proximate composition (dry matter basis) of experimental diets.

Table 2 .
Weight gain (WG), feed conversion ratio (FCR), survival rate (SR), and male frequency (MF) of tilapia at different concentrations of administration and vehicles of 17--methyltestosterone (MT) for 28 days.
Note: 1 Mean (± standard deviation) of four replicates.Values with different superscripts indicate significant differences as determined by Tukey's test (p < 0.05).