In this study, we evaluated the effects of the thawing methodology on sperm function after cryopreservation in pellets. We compared the use of two thawing procedures: method (1) maintaining pellet for 10 min in air at room temperature, then another 10-min period in air at 37°C followed by dilution in a thawing medium; and method (2) immersing the pellets directly in thawing medium at 37°C for 20 min. This procedure leads to a higher rate of temperature increase and a dilution of the glycerol present in the freezing medium. We analyzed the effect of the thawing procedure on sperm motility, viability, membrane lipid packing disorder, acrosome status, reactive oxygen species (ROS) level and sperm chromatin condensation. This study revealed a positive effect of the M2 thawing methodology on sperm parameters. The percentage of spermatozoa with fast-linear movement is increased (M1: 17.26% vs. M2: 28.05%, p<0.01), with higher viability (M1: 37.81% vs. M2: 40.15%, p<0.01) and less acrosome damage (M1: 40.44% vs. M2: 35.45%, p=0.02). We also detected an increase in the percentage of viable spermatozoa with low membrane lipid disorder (M1: 31.36% vs. M2: 33.17%, p=0.03) and a reduction in chromatin condensation (44.62 vs. 46.62 arbitrary units, p=0.02). Further studies will be necessary to evaluate the possible clinical applications.
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