Abstract
Background:
The increasing demand for animal protein in Indonesia underscores the importance of improving productivity in indigenous buffalo breeds, which remain underutilized. Semen quality plays a central role in artificial insemination success, and metabolomic profiling provides opportunities to uncover molecular mechanisms underlying fertility.

Aim:
This study aimed to investigate post-thawed sperm quality and metabolomic signatures in three Indonesian buffalo breeds—Silangit, Toraja, and Sapala to identify breed-specific biomarkers associated with fertility potential.

Methods:
Semen samples were collected from healthy bulls (5–8 years, 450–750 kg) maintained under standardized management conditions. Post-thawed semen quality was evaluated for motility, viability, abnormality, acrosome and membrane integrity, along with kinematic parameters using computer-assisted sperm analysis. Metabolomic profiling was performed with high-resolution LC-HRMS, and data were analyzed using multivariate statistics, pathway enrichment, and receiver operating characteristic (ROC) curve analysis for biomarker evaluation.

Results:
Significant breed-dependent differences were observed in semen quality and metabolomic composition. Silangit bulls showed higher sperm viability and membrane integrity, while Toraja and Sapala bulls exhibited lower abnormality rates. Distinct discriminative metabolites were identified across breeds: oxalacetic acid and adenosine in Silangit; dimethylethanolamine and uridine in Toraja; and glutamic acid with succinic acid semialdehyde in Sapala. Pathway enrichment analysis revealed energy metabolism predominance in Silangit, membrane remodeling and DNA stability in Toraja, and antioxidative mechanisms in Sapala. ROC analysis confirmed the diagnostic potential of these metabolites as candidate biomarkers.

Conclusion:
Integrating semen quality assessments with metabolomic profiling highlighted breed-specific molecular adaptations to cryopreservation in Indonesian buffalo. These findings provide valuable insights into biochemical determinants of sperm function, offering practical opportunities for fertility prediction, improved cryopreservation, and selective breeding to support sustainable utilization of indigenous buffalo genetic resources.

Key words: Fertility prediction; Indigenous genetic resources; Seminal plasma; Metabolomic profiles.