H-Y antigen-based immuno-segregation strategies for bovine X sperm enrichment

Insemination of sperm bearing X and Y chromosomes prior to sexual pre-selection significantly impacts the livestock industry economically. Fluorescence-activated cell sorting (FACS) is a widely accepted technology that sorts sperm with over 90 % accuracy. High costs and potential damage to sperm limit the application of flow cytometry. Consequently, flow cytometry has been replaced by less intrusive and more affordable immunological methods. The present study hypothesizes that antibodies raised against male-enhanced antigen-1 (MEA-1) peptide of Bos indicus will specifically bind to bovine Y sperm, thus assisting in the enrichment of X sperm. A solid surface strategy (SSS) was employed to enrich the X chromosome-bearing spermatozoa of Bos indicus, utilizing purified IgG anti-peptide (MEA P-1) antibodies alongside commercial Human MEA-1 antibodies for comparison. A quantitative SYBR green real-time PCR assay targeting the Y chromosome-specific SRY gene and the X chromosome-specific PLP gene was conducted to determine the ratio of X and Y sperm in the enriched semen samples. The solid surface strategy using commercially available Human MEA-1 antibodies and anti-MEA P-1 antibodies yielded 34.72 ± 6.74 (X: Y; 73:27) and 20.02 ± 4.08 (X: Y; 64:36) % of X sperm enrichment, respectively. The mean percentage of X chromosome content in an unsorted semen sample and cow bull blood was 50.53 ± 0.19 and 49.90 ± 1.27, respectively, aligning with an expected ratio of 1:1. The sperm viability analysis using computer-assisted semen analyzer (CASA) for motility parameters and the hypo-osmotic swelling (HOS) test showed acceptable sperm viability post-treatment with anti-MEA P-1 antibodies. The study evaluated the efficacy and utility of anti-peptide MEA P-1 antibodies as an alternate approach for bovine X sperm enrichment.