Optimizing sperm selection: Effects of microfluidic and colloidal centrifugation processing on DNA fragmentation in thawed stallion sperm

Abstract

Ensuring the integrity of sperm DNA is vital for the success of assisted reproductive technologies (ART) because DNA fragmentation can compromise fertilization outcomes and embryo development. Cryopreservation is a widely used technique for sperm storage; however, the freezing, thawing, and subsequent sperm selection processes can exacerbate DNA damage, presenting significant challenges for its application in ART. Identifying effective sperm selection methods to minimize post-thaw DNA fragmentation is crucial for improving ART success rates. This study evaluated post-thaw sperm DNA fragmentation in cryopreserved sperm from 6 stallions (n = 6) with 2 replicates per stallion. Cryopreserved straws were thawed for 30 s in a water bath set at 37°C and diluted 1:2.5 with Botusemen® Gold (Botupharma USA, Phoenix, AZ, USA) before being processed using either a microfluidic device (VetMotl; VetMotl Inc., Gaithersburg, MD, USA), colloidal centrifugation at 400 × g for 20 min (EquiPure; NidaCon International AB, Sweden), or left unprocessed (control). After undergoing their respective selection methods, all samples were flash-frozen and stored at −80°C until further analysis. Sperm DNA fragmentation was assessed based on the percentage of cells outside the main population (COMPαt) as determined by the Sperm Chromatin Structure Assay (SCSA). Two samples from a fertile control stallion were used to calibrate the flow cytometer for consistent analysis. Analysis was performed by individuals blinded to treatment. Statistical analysis was performed using R (R Core Team, Vienna, Austria, v3.6.0) to fit a mixed linear regression model. The model included treatment method (control, EquiPure, VetMotl) as a fixed effect and stallion as a random effect. No significant differences were observed between stallions (P = 0.074). VetMotl processing resulted in significantly lower (P < 0.001) DNA damage (5.59% ± 1.17) compared with both control (12.95% ± 1.84) and EquiPure (14.61% ± 2.21) treatments. These findings suggest that microfluidic sperm processing may be a promising approach for reducing DNA damage in thawed cryopreserved stallion sperm intended for assisted reproduction.