Differential expression of sperm capacitation-associated microRNAs in fresh and cryopreserved bovine semen

Spermatozoa rely on transcripts acquired during spermatogenesis and post-testicular transit because their DNA is highly condensed. Among these, microRNAs (miRNAs) have emerged as potential regulators of fertility and biomarkers, since they participate in key processes. This study aimed to identify miRNAs associated with sperm capacitation events before and after cryopreservation of bull semen. For this, based on the evaluation of sperm capacitation events (as phosphatidylserine translocation and acrosomal reaction), 12 sexually mature Nellore bulls (3 ejaculates from each) were separated into two groups of bulls with high (HrC, n = 4) and low (LrC, n = 4) responsiveness to in vitro sperm capacitation induction. The sperm miRNA profile was analyzed fresh before (CO) and after capacitation induction (CAP), and after cryopreservation in the HrC and LrC groups. Among the 383 miRNAs evaluated in fresh semen samples, 33 were expressed in both bull groups (HrC and LrC). In CO samples, 19 miRNAs showed differential expression (P ≤ 0.10) between the HrC and LrC, while no difference was observed in CAP samples. In cryopreserved semen, 30 miRNAs were identified, with one miRNA (bta-miR-191) exclusive to the HrC group and two (bta-miR-18a and -106a) exclusive to the LrC. The 19 differentially expressed miRNAs were predicted to regulate 24 signaling pathways and 113 biological processes, with Rap1, VEGF, Relaxin, ErbB, and gap junction being the most significantly enriched KEGG pathways. In conclusion, distinct miRNA profiles associated with sperm capacitation were identified, with bta-miR-191 emerging as a potential biomarker of sperm capacitation in cryopreserved semen.