SignificanceIatrogenic failures of assisted reproduction technology could be associated with routine sperm preparation techniques. Limitations of conventional sperm selection methods include the inability to efficiently sort functional spermatozoa and assess sperm fertilization potential. We developed a robust microfluidic sperm sorting system by using a diffuser-type microfluidic sperm sorter device capable of ultrahigh-throughput selection and separation of motile, DNA-intact, and functionally competent sperm. The strategy inclusively targeted the intrinsic traits related to fertility and successfully produced livebirths from low-dose insemination of microfluidic sorted spermatozoa. The fertile subpopulation was identified based on the kinetic and trajectory patterns as the sinuous, transitional cohort. The clinical significance of microfluidic sperm sorting is reflected by the established pregnancy and live births of calves.

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Background The isolation and characterization of sperm subpopulations that can achieve fertilization is a major challenge of assisted reproduction methods. We focused on the microfluidic sperm sorter as a novel tool for collecting highly motile spermatozoa from heterogeneous semen samples. Objectives This study primarily aims to obtain baseline information on sorted spermatozoa according to its characteristics and in vitro life span. Materials and Methods Frozen‐thawed bull semen was subjected to microfluidic sperm sorting using diffuser‐type microfluidic sperm sorter (DMSS). After sorting, samples were collected as the sorted spermatozoa and unsorted residual spermatozoa and incubated at 37°C for subsequent evaluation. The samples were assessed at different time points (0 or 1, 6, and 24 h) in terms of motility, which was measured by computer‐assisted sperm analysis (CASA), membrane integrity, mitochondrial function, and adenosine triphosphate (ATP) production after sorting (0 h). To determine the characteristics and efficiency of DMSS sorting, the sorted spermatozoa were compared with samples collected using the swim‐up method, a conventional method in motile sperm selection. Results A comparison between the sorted and residual spermatozoa demonstrated significantly higher motility parameters, membrane integrity, and mitochondrial function of the sorted spermatozoa until 6 h after incubation. The time course decrement of membrane and mitochondrial status were subjected to curve fitting and theoretically supported. Sperm ATP production measured immediately after sorting showed higher ATP generation of the sorted spermatozoa compared with the unsorted, frozen‐thawed spermatozoa. The motility parameters and mitochondrial activity of DMSS‐sorted spermatozoa were higher than the swim‐up‐collected spermatozoa (p < 0.05). Discussion and Conclusion These results indicate that DMSS sorting can strictly select highly motile spermatozoa with the ability to maintain its membrane integrity and mitochondrial function related to ATP production. We speculate that the device that is able to sort high‐quality spermatozoa can have great potential in assisted reproduction.

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