Acrosome-Disrupted Dead Sperm Impact the Function of Live Bovine Spermatozoa After Cryopreservation

The influence of dead sperm on their healthy counterparts in bovine semen is not well established. This is particularly relevant to artificial insemination (AI), since semen handling and biotechnological procedures can increase the percentage of dead sperm. This study aimed to evaluate the impact of acrosome-disrupted (sonicated) spermatozoa on the quality of neighbouring untreated viable cells after cryopreservation. Semen samples from 12 healthy Holstein bulls were diluted (80 × 10⁶ sperm/mL) in pre-warmed OptiXcell extender at 38°C. A 6 mL portion of diluted semen underwent sonication, and both sonicated and untreated semen samples were mixed to produce treatment groups (TG) as: TG25%, TG50% and TG75% sonicated sperm. Control (CTRL) was not mixed with sonicated sperm. Progressive sperm motility was assessed during a thermo-resistance test after 30 (on-test) and 120 min (off-test) of incubation at 38°C. Results of delta and relative variation of progressive sperm motility showed a significant decline in the TG75% compared to the CTRL (p = 0.013 and 0.034, respectively). Flow cytometry revealed a gradual decline in percentage of viable acrosome-intact sperm with low membrane fluidity and low intracellular calcium (p < 0.001). A comparable decrease was observed for percentage of viable acrosome-intact sperm with high mitochondrial membrane potential (p < 0.001). Considering these findings, this study suggests that post-sonication leakage of acrosomal/cellular content could compromise the functionality of untreated spermatozoa, highlighting the necessity to conduct further mechanistic investigation to evaluate possible damaging pathways.