Optimization of hormonal dose during masculinization of tilapia ( Oreochromis niloticus ) fry

The experiment was conducted to optimize the dose of 17α-methyl testosterone (MT) during masculinization of tilapia (Oreochromis niloticus) fry. Five treatments were designed with various doses of hormone to find out the most effective one. The treatments were designated as T1 (0 mg MT/kg), T2 (40 mg MT/kg), T3 (50 mg MT/kg), T4 (60 mg MT/kg) and T5 (70 mg MT/kg). MT was administered orally by using nursery feed and Ethanol (as hormone carrier solvents) diet to tilapia fry for 28 days in hapa. After completion of the trial period of 28 days nursing of the experimental fry was continued for further 2 months with commercial feed. At the end of experiment the sex ratio was determined by examining gonad after dissecting the fish. Growth performance was monitored by recording the morphometric characteristics i.e. weight gain (g), average daily weight gain (g). The analysis of growth data showed significant variation in weight, % weight gain, SGR (% /day) of fish among the different treatments. SGR increases with the increase of hormone dose. The present study demonstrated that all MT receiving treatments showed a significantly (p<0.01) higher male proportion (94.28%) than control (48.57%). The dose of 60 mg MT/kg of feed resulted in maximum male population (94.28%). The result indicated that the optimum dose of MT hormone was 60 mg /kg with a feeding period of 28 days after hatching.


Introduction
Tilapia is likely to be the most important of all aquaculture fish in 21 st century.Tilapia has certain favourable characteristics, like tolerant to adverse environmental conditions, can survive at low dissolved O 2, euryhaline, relatively fast growth and efficient food conversion.All these characteristics make tilapia one of the best choices for farmers (Yi et al, 1996;Penna-Mendoza et al, 2005).
Despite having many good characteristics, one of the main impediments in tilapia production at commercial scale is its precocious reproduction.It attains sexual maturity at early age and reproduces after every 4-6 weeks in the pond.The mono-sex culture technique can be used to control this unwanted reproduction of tilapia by culturing all male tilapia in pond.Tilapia has sexual growth dimorphism in which males grow faster and have more standard size than females (Muir and Little, 1991).
There are four strategies for mono-sex male culture i.e. manual process by visual examination; hybridization; gene manipulation and masculinization via steroid hormone.At the time of hatching tilapia fry are sexually undeveloped.Hence, during the early period of gonadal differentiation, changes in sex hormone level can affect the final sex independently of the genetic sex (Andersen et al., 2003).
Hormonal sex reversal is a technique of changing of sexes from one sex to another in fish by administering synthetic steroid hormones before or during the period of sexual differentiation.In this technique, the first feeding fry are treated with male hormone or androgens (17α-methyl testosterone), which develops testes and male sexual characteristics at maturity.Hormone treatment does not alter the genotype of the fish but directs the expression of the phenotype.Hormonal sex reversal quickly became the methodology of choice to produce single sex population.Low dose of hormone, usually 17α-methyl testosterone, added to the early fry diet in between 40-60 ppm, when correctly administered, could guarantee almost complete sex reversal of female tilapia into phenotypic male.In tilapia, sex steroid hormones play a promising role in directing the gonadal differentiation process (Piferrer, 2001).17α-methyltestosterone (MT) is a synthetically produced anabolic and androgenic steroid hormone.This hormone promotes both muscle growth and development of male sexual characters.However, it is important to identify the optimal level of MT as well as duration and timing of treatment for consistent, successful sex reversal (Dunhan, 1990).Tilapia culture is increasing in Bangladesh and farmers need sufficient good quality monosex tilapia.The aim of this study was, therefore, to find out optimum dose rate of MT treatment for sex reversal along with its effect on growth performance of Nile tilapia (Oreochromis niloticus).

Sources of fry and experimental design
Nile tilapia (O.niloticus) fry used in the present experiment were collected from "Reliance Aqua Farm", Boilor, Trishal, Mymensingh.Three days old spawns (first feeding stage) of tilapia (O.niloticus) were stocked in 10 (Ten) hapa (Size 6ft×6ft).

Hormone-feed preparation and feeding trial
Four diets with different doses of MT hormone i.e. 40, 50, 60 and 70 mg/kg were prepared through ethanol evaporation method (Muir and Santiago, 1994).To prepare 5 kg feed for each treatment required amount of MT hormone (i.e.200, 250, 300, 350 mg MT hormone for T 2 , T 3 , T 4 and T 5 respectively) was diluted with 750 ml ethanol carrier for homogenous mixing with the nursery feed.In case of control, only nursery feed was used without hormone treatment.The fry were fed with hormone mixed feed 5 times (started at 8.00 am and continued up to 6.00 pm with two hour interval) a day up to satiation.The hormone feeding phase was continued for a period of 28 days.The fish were then fed with hormone free commercially available diet for further two months.

Growth Parameters
Growth performance was calculated according to the following formulae after Rashid (2008).Weight gain (g) = Mean final weight (g) -mean initial weight (g) % Weight gain = Mean final fish weight-mean initial fish weight Mean initial fish weight × 100 Average daily weight gain (g) = Mean final weight-Mean initial weight

Collection and analysis of data
Ten fry from each of the hapa were randomly selected for measuring weight (g) at an interval of 7 days.At the end of the rearing period, the male and female fish were sexed by gonad squashing and acetocarmine staining method (Guerrero and Shelton, 1974).
The weight gain (g), average daily weight gain (g) and specific growth rate (%/day) of fish during experiment with different hormone feeds were tested using one way analysis of variance (ANOVA).The statistical analysis was performed with the aid of the computer software SPSS programme and MS Excel Programme.

Sex reversal
An objective of this study was to find out the minimum dose of 17-alpha methyl testosterone to obtain all male population of Nile tilapia (Oreochromis niloticus).Results of this study showed that each hormone treated group gave a significantly higher male/female ratio (Table 1), while the control group showed normal male and female ratio.No treatment gave 100% male population of O. niloticus.In this study maximum male population (94.28 %) of Nile tilapia was obtained at the dose of 60 mg MT/kg of feed for 28 days, while the minimum male proportion (88.57%) was recorded for dose of 40 mg MT/kg of feed for 28 days (Table 1, Fig. 1 ).The dose of 50 and 70 mg MT/kg of feed were resulted in 91.43% males (Table 1).Statistical analysis of the sex reversal data showed a highly significant difference (P<0.01) between treatments (Table 1).The percentage of male fish observed all treatments receiving MT showed a greater and significantly higher (p<0.01)male proportion as compared to control treatment.
17α-methyl testosterone was effective in producing phenotypic male of tilapia.Greater than 90% male populations have been obtained at a variety of dose rates.Jae-  Romerio et al. (2000) obtained 98 % male population at the dose of 60 mg MT/kg of feed.Smith and Phelps (2001), reported 99-100 % male Nile tilapia when given MT at 60 mg/kg of feed.The results of this study showed a significantly lower male proportion (91.43 %) for highest dose rate of androgen i.e. 70 mg MT/kg of feed.These results are in line with the findings of Okoko (1996), who obtained 99.3% males at 30 mg MT/kg, while 97 and 71.9% males at the dose rates of 60 and 120 mg MT respectively.He further reported that higher dose rates of MT/kg of feed resulted in no increase of male percentage.
However, duration of treatment for successful sex-reversal of tilapia fry might be an important consideration and the duration used in the present study closely followed the duration of Okoko (1996).
High rate of masculinization in tilapia can be influenced by some important factors like hormone concentration, treatment duration, age and size of fry, availability of natural feed, stocking density and feeding frequency (Muir and Little, 1991).

Growth performance parameters
Different dose of MT, significantly influences the growth of Oreochromis niloticus.All the treatments which received MT, showed more average body weight and gain in body weight of Oreochromis niloticus than the control (Table 2, Fig. 2).Treatment T 5 (70 mg MT /kg) showed 0.27g gain in weight followed by T 4 (60 mg MT/kg) 0.16g, Treatment T 3 (50 mg MT/kg) 0.14g, Treatment T 2 (40 mg MT/kg) 0.12 g and T 1 (control) 0.098g at the end of 28 days.The best percent weight gain (1350%) was observed in case of the higher doses of hormone T 5 (70 mg MT/kg) (Table 2).The specific growth rate (%/day) of Nile tilapia ranged between 6.317 (control) and 9.516 % (70mg MT/kg) during this study (Table 2).Statistical analysis of SGR data revealed that all treatments receiving methyl testosterone showed a significant effect on the specific growth rate of fish.Statistical analysis on gain in body weight, showed a highly significant difference among different sex reversal treatments.Hanson et al. (1983) reported that 10-60 ppm MT-treatment showed the best growth than control.Varadaraj et al. (1994) observed faster growth in O. mossambicus when fed 17-alpha methyl testosterone.These results are also in line with Dan and Little (2000), who compared the culture performance of different strains of O. niloticus and found that considering all strains, MT treatment resulted in a final size of fish larger than mixed sex fish.
The present study indicates that there is a positive relationship between doses of androgen and the percentage of male fish, but higher dose (70 mg/kg) of hormone resulted in no increase of male fish.The result of the present research revealed that the optimum dose of MT hormone was 60 mg/kg with a feeding period of 28 days.The dose of 60 mg 17α-methyl testosterone per kg of feed was found to be best for inducing sex reversal in O. niloticus resulting 94.28% (higher) male in the population.

Fig. 2 .
Fig. 2. Weight (g) of Oreochromis niloticus fry under five different treatments Yoon et al. (1988)obtained 97% O. niloticus males at dose rate of 10 mg MT/ kg of diet.Vera-Cruz and Mair (1994) obtained 95 to 98% males with 40 mg MT/kg of diet and 99% with 60 mg MT/kg of diet fed at 20% body weight for 25 days.