The selection of high-quality spermatozoa is critical in assisted reproductive technologies (ART), particularly when working with sex-sorted semen, which is often associated with reduced viability. This study aimed to compare three sperm selection techniques—SwimUp, Sperm Washing, and a Microfluidic Chip (Yeni Biotechnology Inc, Izmir, Turkey)—in terms of their ability to enhance sperm motility and function in sex-sorted bull semen. All samples originated from a single ejaculate batch of one bull and were processed using three thawed sex-sorted straws (Anadolu Stockbreeding, Turkey). Each technique was repeated ten times (n=10/group). CASA evaluations were conducted using the SCA system (Microptics, Barcelona, Spain), while flow cytometric analyses were performed using the CytoFLEX (Beckman Coulter, Brea, CA, USA) to assess mitochondrial membrane potential (HMMP), membrane integrity (SYBR/PI), acrosome integrity (FITC/PI), capacitation status (CAP) and lipid peroxidation (MLPO). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test (P<0.05). The Chip group demonstrated superior performance, with a mean total motility of 83.1%, progressive motility of 73.9%, and velocity values of VCL: 101.7 µm/s, VSL: 70.6 µm/s, and VAP: 86.4 µm/s. These results were significantly higher than those of the Swim-Up group (69.4%, 46.7%, 74.9, 48.7, and 62.7 µm/s) and the Sperm Washing group (34.5%, 23.5%, 67.4, 44.5, and 53.5 µm/s, respectively). Flow cytometry further confirmed enhanced mitochondrial activity, better acrosomal and membrane integrity, lower oxidative stress, and reduced premature capacitation in the Chip-selected spermatozoa (P<0.05). Despite improved quality, sperm concentration was significantly reduced after chip-based selection compared to the other techniques (P<0.05). In conclusion, microfluidic chip-based sperm selection significantly improves the motility and functional integrity of sexed bull sperm compared to Swim-Up and Sperm Washing methods, offering a robust strategy for optimizing ART outcomes. Further analyses will evaluate the impact of these selection techniques on in vitro embryo production quality and will evaluate semen from more bulls.
Microfluidic chip-based sperm selection improves motility and functional quality of sex-sorted bull semen compared to swim-up and washing techniques
Enrico Dalle Palle;
2025
Abstract
The selection of high-quality spermatozoa is critical in assisted reproductive technologies (ART), particularly when working with sex-sorted semen, which is often associated with reduced viability. This study aimed to compare three sperm selection techniques—SwimUp, Sperm Washing, and a Microfluidic Chip (Yeni Biotechnology Inc, Izmir, Turkey)—in terms of their ability to enhance sperm motility and function in sex-sorted bull semen. All samples originated from a single ejaculate batch of one bull and were processed using three thawed sex-sorted straws (Anadolu Stockbreeding, Turkey). Each technique was repeated ten times (n=10/group). CASA evaluations were conducted using the SCA system (Microptics, Barcelona, Spain), while flow cytometric analyses were performed using the CytoFLEX (Beckman Coulter, Brea, CA, USA) to assess mitochondrial membrane potential (HMMP), membrane integrity (SYBR/PI), acrosome integrity (FITC/PI), capacitation status (CAP) and lipid peroxidation (MLPO). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test (P<0.05). The Chip group demonstrated superior performance, with a mean total motility of 83.1%, progressive motility of 73.9%, and velocity values of VCL: 101.7 µm/s, VSL: 70.6 µm/s, and VAP: 86.4 µm/s. These results were significantly higher than those of the Swim-Up group (69.4%, 46.7%, 74.9, 48.7, and 62.7 µm/s) and the Sperm Washing group (34.5%, 23.5%, 67.4, 44.5, and 53.5 µm/s, respectively). Flow cytometry further confirmed enhanced mitochondrial activity, better acrosomal and membrane integrity, lower oxidative stress, and reduced premature capacitation in the Chip-selected spermatozoa (P<0.05). Despite improved quality, sperm concentration was significantly reduced after chip-based selection compared to the other techniques (P<0.05). In conclusion, microfluidic chip-based sperm selection significantly improves the motility and functional integrity of sexed bull sperm compared to Swim-Up and Sperm Washing methods, offering a robust strategy for optimizing ART outcomes. Further analyses will evaluate the impact of these selection techniques on in vitro embryo production quality and will evaluate semen from more bulls.
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