Introduction: The production of all-female populations is widely used in rainbow trout (Oncorhynchus mykiss) aquaculture to improve growth performance, size uniformity, and economic efficiency. This approach commonly depends on the production of functional XX neomales using 17α-methyltestosterone (17α-MT). However, concerns regarding the environmental persistence and endocrine-disrupting effects of synthetic steroids have increased interest in sustainable alternatives. Plant-derived bioactive compounds have emerged as promising candidates due to their biodegradability and low environmental impact. Among these, Tribulus terrestris contains steroidal saponins and androgen-like compounds with reported effects on growth and reproductive performance. Although its beneficial effects have been documented in several fish species, information on its masculinizing potential during sex differentiation in salmonids remains limited. Therefore, this study evaluated the effectiveness of the hydroalcoholic extract of T. terrestris, administered by immersion during the sex differentiation period, as a natural alternative to 17α-MT for inducing masculinization in rainbow trout.

Material and Methods: The experiment was conducted at the Cold-Water Fish Genetics and Breeding Research Center, Yasuj, Iran, using rainbow trout larvae obtained from pooled gametes of multiple broodstock. A total of 22,500 fertilized eggs were incubated under standard hatchery conditions and randomly assigned to five experimental treatments, each with three replicates. Larvae were exposed by immersion once weekly for seven consecutive weeks during the sex differentiation period to the hydroalcoholic extract of Tribulus terrestris at concentrations of 20, 40, and 80 mL L⁻¹. A positive control group was treated with 17α-methyltestosterone (400 μg L⁻¹), while an untreated group served as the negative control. Each immersion treatment lasted 2 h under continuous aeration at 11–12°C. At the end of the rearing period, the survival rate was recorded, and the sex ratio was determined through gonadal histological examination. Data were analyzed using one-way ANOVA followed by Duncan’s multiple range test at a significance level of P<0.05.

Results and Discussion: The highest masculinization rate was observed in the 17α-methyltestosterone treatment (85.61 ± 1.52%), which was significantly higher than that of the control group (73.48 ± 0.49%) (p<0.05). Among the Tribulus terrestris treatments, only the 20 mL L⁻¹ concentration maintained survival throughout the experimental period. In contrast, complete mortality occurred in the 40 and 80 mL L⁻¹ groups within the first two weeks of exposure. The 20 mL L⁻¹ treatment resulted in a masculinization rate of 68.47±1.18%, which differed significantly from the control group (p<0.05). No significant differences in survival rate were detected among the control, 17α-methyltestosterone, and 20 mL L⁻¹ Tribulus treatments (p<0.05). Histological examination revealed no intersex individuals in any treatment group. Furthermore, testes from fish treated with 20 mL L⁻¹ Tribulus extract exhibited normal gonadal architecture with well-developed spermatogenic cells and no evidence of tissue abnormalities or structural damage.

Conclusion: The results demonstrated that the hydroalcoholic extract of Tribulus terrestris at 20 mL L⁻¹ significantly increased the proportion of males in rainbow trout without adversely affecting survival or gonadal structure. Although the masculinization efficiency of 17α-methyltestosterone remained higher, the observed effect of Tribulus extract suggests its potential as a natural sex-control agent in salmonid aquaculture. The complete mortality observed at higher concentrations (40 and 80 mL L⁻¹) highlights the importance of dose optimization when applying phytogenic compounds. Histological findings confirmed normal testicular development and the absence of tissue abnormalities or intersex individuals in fish treated with the effective dose. These results indicate that T. terrestris may influence sex differentiation pathways while maintaining gonadal integrity. Overall, the hydroalcoholic extract of T. terrestris can be considered a promising environmentally friendly alternative for increasing male proportion in rainbow trout; however, further molecular and long-term studies are required to clarify its mechanism of action and optimize its practical application in aquaculture.