High-Efficiency Bovine Sperm Sexing Used Magnetic-Activated Cell Sorting by Coupling scFv Antibodies Specific to Y-Chromosome-Bearing Sperm on Magnetic Microbeads

SIMPLE SUMMARY: Female calves are favored for milk production and genetic advancement in the dairy industry, and sex selection by using sexed semen has been long considered. A potential alternative sperm sexing technique is magnetic-activated cell sorting combined with an immunological method that uses scFv antibodies against male-specific sites on Y-chromosome-bearing sperm; however, the technique should be evaluated for validity and accuracy. This study focuses on how well bovine sperm are separated by the use of magnetic microbeads coupled with scFv antibodies against Y-chromosome-bearing sperm (PY-microbeads). The results showed that sexed bovine sperm using PY-microbeads was a highly effective technique for distinguishing X- and Y-chromosome-bearing sperm. It had no negative impact on the quality of X-chromosome-bearing sperm. The technique produced 82.65% of X-chromosome sperm in the X-enriched fraction semen and 81.43% of Y-chromosome sperm in the Y-enriched fraction semen, which was utilized to generate target sexed bovine semen. ABSTRACT: Sperm sexing technique is favored in the dairy industry. This research focuses on the efficiency of bovine sperm sexing using magnetic-activated cell sorting (MACS) by scFv antibody against Y-chromosome-bearing sperm (Y-scFv) coupled to magnetic microbeads and its effects on kinematic variables, sperm quality, and X/Y-sperm ratio. In this study, the optimal concentration of Y-scFv antibody coupling to the surface of magnetic microbeads was 2–4 mg/mL. PY-microbeads revealed significantly enriched Y-chromosome-bearing sperm (Y-sperm) in the eluted fraction (78.01–81.43%) and X-chromosome-bearing sperm (X-sperm) in the supernatant fraction (79.04–82.65%). The quality of frozen–thawed sexed sperm was analyzed by CASA and imaging flow cytometer, which showed that PY-microbeads did not have a negative effect on X-sperm motility, viability, or acrosome integrity. However, sexed Y-sperm had significantly decreased motility and viability. The X/Y-sperm ratio was determined using an imaging flow cytometer and real-time PCR. PY-microbeads produced sperm with up to 82.65% X-sperm in the X-enriched fraction and up to 81.43% Y-sperm in the Y-enriched fraction. Bovine sperm sexing by PY-microbeads showed high efficiency in separating Y-sperm from X-sperm and acceptable sperm quality. This initial technique is feasible for bovine sperm sexing, which increases the number of heifers in dairy herds while lowering production expenses.