135 Vortex-based thawing of pelleted frozen porcine semen prepared for IVF: a CASA and flow cytometry analysis

Commercial porcine semen is commonly frozen in pellets or 5-mL straws. Pelleted frozen semen is a convenient presentation for handling and splitting frozen porcine semen doses for use in IVF. This study aimed to compare two thawing methods (vortex vs. hand-shake-based) combined with two sperm processing methods (colloid centrifugation and direct resuspension of sperm in IVF medium vs. the inclusion of a sperm rinse step) and evaluate which treatment resulted in improved motility and sperm characteristics. Thus, the study consisted of four treatments: Trt1, hand-shake thawing combined with a sperm rinse step following colloid centrifugation; Trt2, vortex-based thawing combined with a sperm rinse step following colloid centrifugation; Trt3, hand-shake thawing combined with direct sperm resuspension in IVF medium following colloid centrifugation; and Trt4, vortex-based thawing combined with direct sperm resuspension in IVF medium following colloid centrifugation. Four boars were evaluated (four repetitions per boar). The thawing procedure was as follows: one pellet of frozen semen was transferred from liquid nitrogen to a 5-mL centrifuge tube containing 1 mL of porcine semen pellet thawing solution at 48°C and vigorously hand-shaked or vortexed for 7 s. The thawed semen was laid over 1 mL of one-layer colloid solution (Equipure) at 37°C and centrifuged at 1000g for 10 min. Following colloid centrifugation, the supernatant was removed and, depending on the treatment, the sperm pellet was either directly resuspended in 300 µL of IVF medium (Porcine Fertilization Medium) or subjected to a sperm rinse centrifugation step before resuspension in IVF medium. The sperm rinse step consisted in sperm pellet resuspension in 1 mL of sperm rinse solution and centrifugation at 500g for 5 min. Following the sperm rinse step, the supernatant was removed and the sperm pellet was resuspended in 300 µL of IVF medium. In all treatments, the sperm in IVF medium was incubated in 5% CO₂ at 38°C for 1 h. Sperm motility and viability, acrosome membrane integrity, and sperm agglutination were evaluated following incubation. Motility was analyzed through computer-assisted sperm analysis (CASA). Sperm agglutination was evaluated through visual sperm counting using pictures taken by the CASA system. At least five pictures per treatment per replicate containing 70–100 sperm per picture were analyzed. Flow cytometry was used to evaluate viability (live/dead stain kit) and acrosome membrane integrity (FITC-PNA). Results of the experiment were analyzed through ANOVA and Tukey test (SAS 9.4). Percentage results were subjected to ArcSin Square Root transformation. There was no difference between treatments in total or progressive motility (P > 0.05). Similarly, there was no difference between treatments when evaluating viability and acrosome membrane integrity (P > 0.05). Importantly, Trt4 resulted in significantly less sperm agglutination compared with other treatments (P < 0.001). Vortex-based thawing of pelleted frozen porcine semen without a sperm rinse centrifugation step during sperm processing for IVF resulted in reduced sperm agglutination and might result in improved fertilization and IVF outcomes.