Dietary pine pollen induces masculinization in Nile tilapia (Oreochromis niloticus, L. 1758) by modulating sex-biased gene expression and steroid hormone profiles.

Abstract

Background: The health and environmental risks associated with using 17-methyltestosterone hormone (MT) to produce all-male tilapia stocks limit the sustainability of aquaculture. As such, plant-based products with the potential to induce masculinisation in tilapia are gaining considerable attention as potentially safe, nature-based alternatives. However, the underlying mechanism for sex change induction by plant products remains unknown. The present study used pine pollen (PP) to study the processes modulating female-to-male sex change in three-day-old all-female Nile tilapia (Oreochromis niloticus).

Methods: Experimental fish were fed either a basal diet supplemented with 1,280 mg PP kg^- 1 from 3 to 30 days post-hatch (dph), or the same basal diet incorporated with 60 mg MT kg^- 1 (MT treatment), or the basal diet only (CT treatment. After 30 dph, fish in all treatments were fed only a basal diet up to 120 dph.

Results: Histological observations showed that female-to-male sex change occurred between 21 and 45 dph in MT and PP treatments. During this period, gonadal tissue in PP-treated fish progressively shifted from undifferentiated germ cells toward testes dominated by spermatogonia and spermatocytes. In contrast, MT treatment accelerated spermatogenic progression, with gonads exhibiting advanced germ cells, including spermatids and spermatozoa, reflecting complete masculinisation. Conversely, fish in the CT group underwent normal ovarian differentiation between 21 and 30 dph, characterised by ovarian cavity formation and oocyte-like germ cells. These gonadal histological changes were closely related to temporal changes in sex-biased gene expression and steroid hormone profiles. The PP and MT treatments significantly up-regulated male sex gene expression (dmrt1 and amh) and down-regulated female transcripts (cyp19a1a and foxl2), whereas an opposite pattern was observed in the CT group. Correspondingly, sex steroid concentrations (testosterone and 11-ketotestosterone) were also significantly elevated in both PP- and MT-treated fish, consistent with higher proportions of male individuals (PP: 77.8 ± 2.9% and MT: 97.8 ± 1.1%), unlike the CT group, where fish remained all-female with significantly higher 17β-estradiol levels.

Conclusion: These findings imply that PP induces female-to-male sex change in Nile tilapia by disrupting the expression of sex-biased genes and, consequently, the androgen-to-estrogen balance. However, further studies are required to enhance the androgenic potency of PP.