Comparison of Post-Thaw Motility and In Vitro Fertility Between Ejaculated and Epididymal Semen, and Seminal cfDNA Characterization in Pantaneiro Bulls

This study evaluated the post-thaw motility and in vitro fertility of ejaculated and epididymal semen from Pantaneiro bulls and characterized cell-free DNA (cfDNA) in fresh seminal plasma. Semen from five bulls was collected via electroejaculation or post-mortem epididymal extraction. Fresh semen parameters and cfDNA concentrations were assessed before cryopreservation. Post-thaw sperm kinetics were evaluated using CASA at 0 and 6 h of incubation, and in vitro embryo development was analyzed following IVF. Data were assessed using ANOVA and logistic regression. Ejaculate samples exhibited more morphological defects than epididymal samples (15.8% vs. 1.8%, <i>p</i> ≤ 0.05). Post-thaw, epididymal semen showed higher total (87.2% vs. 32.4%) and progressive (67.1% vs. 14.4%) motility at 0 h (<i>p</i> ≤ 0.05), and higher motility at 6 h (38.9% vs. 11.0%, <i>p</i> ≤ 0.05). In vitro fertility did not differ significantly between ejaculated (n = 525 oocytes) and epididymal (n = 500 oocytes) semen groups in terms of cleavage (49.6% vs. 44.2%) and blastocyst formation on D7 (26.1% vs. 22.2%, <i>p</i> > 0.05). cfDNA concentration in fresh semen ranged from 11.4 to 50.9 ng/µL. These findings indicate that epididymal sperm from Pantaneiro bulls retain high post-thaw motility and fertility. Additionally, cfDNA characterization in seminal plasma contributes to indigenous cattle preservation and advances in male fertility research.