Chaperoning post-thaw cryo-competence: HSP90 abundance as a molecular signature of sperm quality, oxidative status, cellular integrity, and fertility potential in Indonesian dairy bulls

Improving reproductive efficiency in dairy cattle is critical for sustainable artificial insemination (AI)-based breeding systems in Indonesia. Variability in semen freezability and the limited predictive value of conventional sperm assessments remain challenges for fertility evaluation. This study investigated Heat Shock Protein 90 (HSP90) as a molecular marker associated with sperm cryotolerance and fertility in Indonesian Holstein-Friesian bulls. Cryopreserved semen from 20 bulls was classified into good freezability (GF) and poor freezability (PF) groups based on post-thaw sperm plasma membrane integrity, determined using SYBR-14/propidium iodide (PI) double staining and flow cytometry. Sperm quality, oxidative stress markers (malondialdehyde, MDA; aspartate aminotransferase, AST), HSP90 mRNA and protein abundance, and field fertility (first-service conception rate) were evaluated. GF bulls exhibited higher HSP90 mRNA (0.068 ± 0.003 vs. 0.018 ± 0.003) and protein abundance (0.72 ± 0.02 vs. 0.41 ± 0.02 ng/mL), together with improved motility, kinematic parameters, acrosome integrity, and mitochondrial membrane potential and fewer sperm abnormalities than PF bulls (P < 0.05). PF bulls showed higher MDA and AST levels (P < 0.05). HSP90 expression was strongly correlated with fertility rate (r = 0.964 for mRNA; r = 0.945 for protein) and sperm functional parameters (r ≈ 0.95-0.99), while negative correlations were observed with MDA, AST, and sperm defects (r ≈ -0.92 to -0.98). Multivariable regression models were highly significant (P < 0.001), with post-thaw sperm quality and fertility-related parameters collectively explaining 98.4% and 99.2% of the variation in HSP90 mRNA and protein expression, respectively. Overall, HSP90 abundance was strongly associated with post-thaw sperm quality, biochemical indicators of cellular damage, and field fertility, supporting its potential as a candidate molecular biomarker for evaluating post-thaw sperm quality and reproductive performance in Indonesian dairy bulls. Further validation using paired pre-freezing and post-thaw assessments is needed to determine whether HSP90 abundance is associated with the preservation of sperm quality during cryopreservation.