Abstract
Donggala cattle (Bos indicus), an indigenous Indonesian breed, possess strong adaptability to tropical stressors and favourable reproductive traits, making them a strategic focus for genetic improvement via artificial insemination (AI). However, optimizing AI success is constrained by variable semen quality following cryopreservation. This research examined the relationship between the abundance of heat shock protein 70 (HSP70) at both the mRNA and protein levels and post-thaw sperm quality, aiming to assess its viability as a molecular marker of motility in Donggala bulls. A total of six Donggala bulls were included in this study, representing the entire available population at the study site. Based on progressive motility evaluated using computer-assisted sperm analysis (CASA), the bulls were classified into two groups: good motility and poor motility. Comprehensive semen quality assessments were conducted, including viability, plasma membrane and acrosome integrity, DNA fragmentation, and protamine deficiency. HSP70 abundance at both the transcript (RT-qPCR) and protein (enzyme immunoassay) levels was quantified in cryopreserved sperm. Bulls in the good-motility group demonstrated significantly higher values for progressive and total motility, viability, plasma membrane and acrosome integrity, and DNA integrity ( P < 0.05 ), as well as elevated HSP70 mRNA and protein abundance. Correlation analyses revealed strong positive associations between HSP70 expression and key sperm quality parameters, particularly progressive motility ( R2 > 0.86 ) and acrosome integrity ( R2 > 0.67 ). These findings suggest that HSP70 enhances sperm resilience to cryopreservation-induced oxidative stress by stabilizing membranes, proteins, and DNA. This study is the first to characterize HSP70 abundance in Donggala bulls, providing foundational evidence of its utility as a motility biomarker. Incorporating HSP70 profiling into sire selection may improve AI outcomes and support the sustainable propagation of this valuable native breed.