## 4 Discussion  
Indirect processes, which include feminizing by synthetic sex steroids have generally been used to produce all-female stocks, but they have some problems, such as high costs and toxicity. Hence, the main purpose of this study was the advance of indirect techniques for the production of all-female populations of _Danio rerio_ through the use of natural steroids. Therefore, _Vitex agnus-castus_ hydroalcoholic extract was used as a natural steroid. The present research showed that _Vitex agnus-castus_ extract was effective in successful growth performance in Zebrafish. Therefore, it appears that _Vitex agnus-castus_ could proficiently be used as a natural alternative to synthetic hormones for sex reversal in aquaculture industry. The results achieved confirmed that the effect of _Vitex agnus-castus_ was not significant on the survival rate (\(p>0.05\)), but growth indices were affected meaningfully with the increasing of _Vitex agnus-castus_ doses (\(p<0.05\)). The findings in this research were in agreement with the results of [PERSON] and [PERSON], (2005) [36] on African catfish, [PERSON] and [PERSON] (2015) [37] on goldfish and [PERSON] et al. (2014) [38] on _Cyprinus carpio_. They reported that growth performance was positively affected by herbal extract in the food. According to the significant differences among the treatments of the control group and experimental groups in terms of growth performance, it can be concluded that the _Vitex agnus-castus_ has beneficial and nutrient materials that can positively affect growth indices. Constructed on the present study, _Vitex agmus-castus_ extract (15 g kg\({}^{-1}\) food) was most effective infertility and weight gain (Table 3). With esteem to determining the optimum concentration for growing the growth performance and feminization of Zebrafish life stage in this revision, it was found that the level of 15 g\({}^{-1}\) kg food is suggested as the best concentration. It has been stated that gonad development in mice was significantly affected by injection or oral additive _Vitex agmus-castus_ extract among herbal extract effects [39; 40], but insufficient studies have been carried out to estimate the herbal extract on fish gonad development. Microscopic studies showed that _Vitex agmus-castus_ extract influenced the maturity in a shorter time. Therefore, the treatment groups (especially in group T3) were incomparable with the control group, where the maturation of fish was earlier and with more quantity of egg (Figure 1). Subsequently, the treatment group T3 was matured 15 days earlier than the control group. The increase in fertility rate in the test groups may be due to the increase in the level of the estrogenic compound, which could be caused by the action of the extract in the nourishment. Thus, it is essential to use the phytoestrogens such as the _Vitex agmus-castus_ extract before the beginning of the reproduction season. Findings in the present study are consistent with the findings of [PERSON] and [PERSON] (2015) [41]. Their research on _Poecilia reticulata_ indicates that _Femel Foeniculum vulgar_ extract influenced by the maturity of the ovum in a shorter time. Thus, the level of the estrogenic hormone was increased in treatment groups during the experiment by feeding. An increase in Fennel extract in the diet enhanced reproductive activity and the offspring was born in a shorter time. Oocyte diameters were observed between 0.54-0.75 mm, that highest was in T3. According to the results, treatment T3 for precocious puberty in zebrafish is suggested as model species in aquaculture. Also, reproductive performance was significantly better in T3 than the other treatments (\(p<0.05\)). [PERSON] and [PERSON] (2015) [41] described that Guppy larvae (_Poecilia reticulata_) were positively affected by phytoestrogens. The larvae attained the best enactment and displayed the fastest development of gonad using the herbal extract. [PERSON] et al. (2006) [42] reached maximum growth and oocyte maturation of _Epinephus Coiodes_ larvae fed with phytoestrogen.