Age leaves a molecular mark: altered A-Kinase Anchor Protein 4 (proAKAP4) abundance and sperm cell quality drive fertility decline in Bali bulls (Bos sondaicus)

Reproductive performance in bulls is strongly influenced by age-related factors, which can lead to functional and molecular deterioration of sperm even when conventional semen parameters remain within acceptable ranges. This study aimed to evaluate the impact of the age factor on sperm quality and fertility in Bali bulls (Bos sondaicus), with particular emphasis on proAKAP4, the precursor of A-Kinase Anchor Protein 4, an essential regulator of flagellar structure and motility. Seventeen Bali bulls routinely used in artificial insemination (AI) programs were classified into two age categories: mature bulls (≤9 years; n = 8) and older-age bulls (≥11 years; n = 9). Post-thaw semen samples were evaluated for progressive and total motility, sperm kinematics, viability, morphological abnormalities, DNA fragmentation, and protamine deficiency. ProAKAP4 concentrations were quantified using ELISA. Field fertility outcomes were derived from >10,000 AI records collected over two years. Compared with mature bulls, older-age bulls exhibited significantly lower (P < 0.05) motility, viability, and kinematic parameters, alongside higher rates of morphological abnormalities, DNA fragmentation, and protamine deficiency. ProAKAP4 abundance was markedly reduced in older-age bulls and closely mirrored their lower fertility performance. ProAKAP4 levels were positively correlated with motility, viability, and fertility rate, and negatively correlated with morphological and nuclear defects (P < 0.05). In conclusion, the age factor plays a critical role in determining sperm quality and fertility potential in Bali bulls, with proAKAP4 emerging as a sensitive molecular biomarker of sperm function and field fertility. Integrating proAKAP4 quantification into routine semen evaluation may enhance sire selection accuracy and overall reproductive efficiency in AI programs. Considering the bull's age is therefore essential, as it directly influences proAKAP4 abundance, sperm functional integrity, and fertility outcomes.