## 1 Introduction  
The use of zebrafish (_Danio rerio_) is growing rapidly as a particular species in aquaculture research. It has mainly became popular as a model of vertebrate progress because zebrafish embryos grow quickly and are visible. It can be motivated to breed during the year, and growth from a fertilized egg to breeding stage takes only about 3-4 months in the laboratory [1; 2; 3; 4; 5]. Their short reproduction period (three months) makes them a model candidate for histology and genetic researches [6; 7]. There is not much literature in cases of _Vitex agnus-castus_ extract on the gonad morphology for zebrafish. [PERSON](2000) reported that zebrafish has ovary-like gonads during initial life-time and the bisexual difference happening through the development.  
In recent years, the scientific consideration in phytoestrogens has grown significantly, especially in the medical field [8; 9]. These plant combinations are comparable to animal estrogens [10], which are commonly used in aquaculture, and they also have excessive costs compared to plant extracts and their use requires expert labor. Additionally, synthetic hormones have the potential to gather in the aquatic environment [11]. The usage of herbal medicine is a new and effective approach in the aquaculture industry [12; 13; 14; 15].  
_Vitex agmus-castus_ (Verbenaceae) is a phytoestrogen herb; therefore, it has medical usage in different countries. It is a small shrub native to European, Mediterranean, and Central Asian countries. Also, it has been long used traditionally for its medicinal properties for female reproduction conditions. Phytoestrogens are similar to 17 \(\beta\)-estradiol, in terms of the structure or properties and function [16; 17]. Vitex, systematically known as _Vitex agmus-castus_, belongs to the Verbenaceae family [18]. All parts of this plant have a different kind of essence among which sabinene, cineol, sesquiterpene, and pinene. Essential oils are other components of this plant [19; 20]. _Vitex agmus-castus_ is of the main plants used in herbal medicine for regulating hormones and women's hormonal sicknesses [21]. It is mainly used to treat breast maladies, menstrual irregularities and uterine bleeding [22]. Other properties discovered in Iranian traditional medicine for _Vitex agmus-castus_, include anti-inflammatory, nutritious, aphrodisiac, diuretic, appetizing, carminative, relaxing and anti-flatulence properties. This herb is one of phytoestrogen. Most of the phytoestrogens-based provisions are easily available on the market, and are suggested as legal and non-toxic anti-oestrogenic compounds (to counterbalance the side effects of anabolic steroids, mainly based on methoxyflurane [23], as sexual enhancing compounds based mainly on plant extracts such as _Vitex agmus-castus_ (isoflavones). _Vitex agmus-castus_ phytoestrogen contains isoflavues with estrogenic activity.  
[PERSON] et al., (2002) [24] indicated that phytoestrogens could play a main role in the reproductive process, like estrogens. There are numerous studies associated with subjects, involving the properties of varied hormones on reproduction physiology, morphological reproductive toxicology and development of the ovaries in Zebrafish [25; 26; 27]. In contrast, there are not numerous studies using the phytoestrogen in the gonad development and sex ratio of this species in the literature. This is the first report regarding the potential of _Vitex agmus-castus_ as a feminization agent in cultured food fish. Therefore, this study was conducted to consider the effects of diverse levels of _Vitex agmus-castus_ hydroalcoholic extract on the sex ratio, histology and growth indices of the gonad in zebrafish (_Danio rerio_).