Mineral-driven molecular signatures of energy metabolism underpin sperm motility in buffalo

The success of spermatogenesis depends on the interplay of various biomolecules that ultimately determine sperm quality. In this study, RNA-seq analysis of frozen-thawed buffalo sperm (n = 8) revealed the presence of 263 mineral-associated genes (>1 FPKM) in high (n = 4) and 181 in low motile (n = 4) sperm groups. Among these, 177 mineral-associated genes were commonly expressed between them, and the majority were upregulated (>1 fold), LOC102391588 (ncRNA; 37-fold), ZNF699 (26.5-fold), MYZAP (13-fold), etc., in the high motile group. The expression of selected mineral-associated genes was validated. The top enriched functions in commonly expressed genes were regulation of transcription by RNA polymerase II (FDR: 4.7 × 10⁻²; ZNF331, ZNF692, ZNF180, etc.), followed by spermatogenesis (FDR: 2.9 × 10⁻²; CALR3, ADAM18, ADAM²9, etc.), proton transmembrane transport (FDR: 4.0 × 10⁻²; ATP6V0E1, ATP1A4, ATP6V1B2, etc.) and flagellated sperm motility (FDR: 2.3 × 10⁻¹; CATSPERD, EFCAB6, CABS1, etc.). Additionally, the chromatin remodeling pathway (FDR: 3 × 10⁻²; PTP4A1, PPM1A, DUSP1, etc.) emerged as the most significant and may suggest that these minerals influence genome packaging and sperm functionality. Mineral-associated genes were predominantly associated with zinc (49%), followed by calcium (20%), phosphorus (10%), iron (5%), sodium (2%), potassium (2%), copper (1%) and other trace elements (11%). Although the current study uses frozen-thawed sperm, the findings indicate that mineral-associated genes are crucial for promoting membrane stability, energy production, motility and chromatin integrity, which may contribute to the superior fertilizing ability of sperm.