Molecular Mechanisms Underlying α-Linolenic Acid-Enhanced Reproductive Performance in Male Blue Foxes

α-Linolenic acid (ALA), an essential n-3 polyunsaturated fatty acid, exerts critical effects on animal reproductive function. However, the molecular mechanisms by which dietary ALA regulates reproductive performance in male blue foxes remain largely unclear. In the present study, 16 male blue foxes of similar age and condition were assigned to four dietary groups (n = 4 per group) receiving a basal diet supplemented with 0, 0.5, 1.25, or 2 g/kg of ALA. q-PCR validation further confirmed that medium-dose ALA supplementation significantly upregulated mRNA levels of steroid synthesis-related genes (StAR, LHCGR, INSL3) and antioxidant gene GPX3, while inhibiting the calcium-signaling gene GRIN1. Untargeted liquid chromatography–mass spectrometry (LC–MS) metabolomics of seminal plasma and RNA-seq transcriptomics of testicular tissue were combined to explore the underlying molecular pathways of ALA in terms of its modulating male blue fox reproduction. A total of eight significantly altered seminal plasma metabolites were screened out through metabolomics: mainly arachidonic acid, anandamide, oxaloacetic acid, guanine, lactic acid, dihydrodaidzein, stearic acid, and valylisoleucine. Transcriptomic analysis identified seven differentially expressed genes (DEGs), including FRAS1, COL1A1, CDH17, CLDN4, CTH, KYAT1, and GRIN1, enriched in three core KEGG pathways: ECM-receptor interaction, cell-adhesion molecules and selenocompound metabolism. Spearman correlation analysis revealed extensive significant correlations between seminal-plasma differential metabolites and testicular genes, indicating complicated metabolite-gene crosstalk. Collectively, appropriate dietary ALA remodels the seminal-plasma metabolic microenvironment, stabilizes testicular tissue structure, enhances gonadal antioxidant capacity and facilitates testicular steroidogenesis, thereby improving the reproductive potential of male blue foxes. This study elucidates the nutritional regulatory mechanism of ALA in breeding male blue foxes and provides theoretical references for the application of ALA as a functional feed additive in the production of canines.