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With the advent of sperm sex sorting methods and computer-aided sperm analysis platforms, comparative 2D motility studies showed that there is no significant difference in the swimming speeds of X-sorted and Y-sorted sperm cells, clarifying earlier misconceptions. However, other differences in their swimming dynamics might have been undetectable as conventional optical microscopes are limited in revealing the complete 3D motion of free-swimming sperm cells, due to poor depth resolution and the trade-off between field-of-view and spatial resolution. Using a dual-view on-chip holographic microscope, we acquired the full 3D locomotion of 235X-sorted and 289 Y-sorted bovine sperms, precisely revealing their 3D translational head motion and the angular velocity of their head spin as well as the 3D flagellar motion. Our results confirmed that various motility parameters remain similar between X- and Y-sorted sperm populations; however, we found out that there is a statistically significant difference in Y-sorted bovine sperms’ preference for helix-shaped 3D swimming trajectories, also exhibiting an increased linearity compared to X-sorted sperms. Further research on e.g., the differences in the kinematic response of X-sorted and Y-sorted sperm cells to the surrounding chemicals and ions might shed more light on the origins of these results.

3D imaging of sex-sorted bovine spermatozoon locomotion, head spin and flagellum beating