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

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.

Production of single-chain fragment variable (scFv) antibodies specific to plasma membrane epitopes on bull Y-bearing sperm.

Distinguishing between bull Y- and X-bearing sperm populations is advantageous for techniques using sexed bull semen. The aim of this study was to produce a single-chain fragment variable (scFv) antibody against plasma membrane epitopes on bull Y-bearing sperm. Variable heavy (VH)- and variable light (VL)-region genes generated from a hybridoma cell secreting a specific Y-bearing sperm monoclonal antibody (mAb-1F9) were cloned and expressed. The expected sizes of the DNA bands were ∼350 bp for the VH gene and ∼318 bp for the VL gene. The VH and VL genes were generated and used to construct an scFv gene (∼650 bp), which was expressed in E.coli TG1 cells and produced the corresponding soluble scFv antibody. Compared with the parent mAb-1F9, the scFv antibodies presented a high affinity for Y-bearing sperm and low cross-reactivity with X-bearing sperm. An immunofluorescence analysis confirmed that the scFv antibodies and mAb-1F9 recognize epitopes on the Y-bearing sperm surface. The fluorescence signal was strong on the plasma membrane of Y-bearing sperm but very weak for X-bearing sperm. This study aids the application and production of engineered scFv antibodies specific to Y-bearing sperm to distinguish between Y- and X-bearing sperm populations for techniques involving sexed bull semen.

Spermatological parameters of immunologically sexed bull semen assessed by imaging flow cytometry, and dairy farm trial.

This study compared the quality parameters of bull semen sexed using an immunological method with those of conventional semen by imaging flow cytometry and applied this semen in dairy farm trials. Semen samples were collected from ten ejaculates from five bulls. Each sample was divided into two treatments: conventional semen (CON) and semen sexed using monoclonal male-specific antibodies combined with the complement system for cytotoxicity reaction (IC-sexed). After obtaining frozen-thawed semen, we used imaging flow cytometry to assess acrosome integrity, sperm sex ratio and viability. Sperm morphology was evaluated using eosin-nigrosin staining. The percentage acrosome integrity did not differ between IC-sexed and CON semen (P = 0.313). The sperm sex ratio showed that the percentage of live X-chromosome-bearing sperm was higher than that of live Y-chromosome-bearing sperm in IC-sexed semen (P = 0.001). IC-sexed semen showed a higher percentage of head and tail defects than did CON semen (P = 0.019). In field trials, 585 cows were subjected randomly to AI with CON or IC-sexed semen. The pregnancy rate of the IC-sexed group did not differ from that of the CON group (P = 0.535). However, IC-sexed semen produced a significantly higher percentage of female calves than did CON semen (P = 0.031). Thus, immunological sexing did not adversely affect the acrosome integrity of sperm. Furthermore, a female calf birth rate of over 74 % can potentially be achieved using IC-sexed semen. These findings could help farmers to replace heifers in their herds.