Differential expression patterns of selective energy metabolism associated genes in fresh and freeze-thawed bubaline spermatozoa at uniform durations of in vitro capacitation.

The present study investigated the differential mRNA expression of energy metabolism and stress-response genes (ADCY10, AK1, ATP5F1B, and HSP70) in fresh and frozen-thawed Murrah buffalo spermatozoa subjected to uniform 2-h in vitro capacitation. Semen samples (n = 32 per group) from eight bulls were divided into four groups: fresh non-capacitated (G1, control), fresh capacitated (G2), frozen non-capacitated (G3), and frozen capacitated (G4). Relative expression was analyzed by RT-qPCR using GLUT5 as the reference gene. Results revealed that ADCY10 was significantly upregulated in G2, G3, and G4 compared to G1, with the highest expression in G3 (frozen non-capacitated), indicating cryopreservation-induced premature activation stronger than bicarbonate-driven capacitation. AK1 was exclusively and significantly upregulated only in fresh capacitated spermatozoa (G2), suggesting it as a specific marker of successful physiological capacitation. ATP5F1B showed significant upregulation in both G2 and G3, reflecting high energy demand in true capacitation and metabolic surge post-thawing, but declined in G4. HSP70 was significantly downregulated in all treated groups compared to G1, confirming cryoinjury-related transcript consumption. These findings demonstrate that cryopreservation triggers partial cryo-capacitation with dysregulated energy gene expression, uncoupling true capacitation pathways and contributing to reduced post-thaw fertility in buffaloes.