Integrated brain and testis transcriptomic analyses reveal the benefits of sandy environmental enrichment in Senegalese sole male (Solea senegalensis) reproduction

This study investigates the effects of physical enrichment (sand substrate) on the gonadal development and transcriptomics of juvenile Senegalese sole ( Solea senegalensis ). A total of 1500 fish were distributed into six outdoor fiberglass tanks, three of which included a 2 cm layer of sand to simulate physical enrichment. Throughout a two-year study, the fish were sampled at four intervals, corresponding to developmental stages ranging from juvenile, pre-pubertal to adult. Blood, testis, and brain tissues were collected at each sampling point to analyse hormonal profiles (estradiol, testosterone, follicle-stimulating and luteinizing hormones) and gene expression. Histological analysis of testicular tissue was used to assess the germ cell development stages, while RNA sequencing (RNA-seq) was performed to evaluate the impact of physical enrichment on transcriptomic profiles in both the brain and testes. Sandy environmental enrichment is associated with increased testosterone and luteinizing hormone levels. Bioinformatics analysis identified differentially expressed genes (DETs) associated with reproduction, immune response, and metabolism. GO and KEGG enrichment analysis demonstrated that important terms and pathways associated with the process of spermatogenesis, cognition and metabolism where differentially enriched between males maintained in sand and fiberglass. These results provided new insights into how environmental enrichment influences reproductive physiology and molecular mechanisms in cultured Senegalese sole from very early stages. • Environmental enrichment with sand substrate enhances testis development in sole. • Sand exposure raises testosterone and luteinizing hormone levels in juvenile males. • Transcriptomic output shows genes linked to reproduction, immunity, and metabolism. • Sandy environment modulate spermatogenesis-related pathways in brain and testes. • Early-life habitat complexity enhances welfare and reproduction in Senegalese sole.