Aptamer-based selective enrichment of X and Y sperm: A new frontier in livestock breeding.

Sperm sexing, separation of X- and Y- sperm, has long been a priority in livestock breeding to selectively enhance female or male offspring production. While flow cytometry remains the only validated technique, its low fertilization efficiency limits widespread adoption. Aptamers, single-stranded oligonucleotides with high specificity and affinity, offer a promising alternative for sperm sexing by targeting distinct surface markers on X- or Y-bearing sperm. In this study, we generated aptamers specific to X and Y sperm and employed a streptavidin-based magnetic separation method for sperm enrichment. After incubating mixed semen samples with the aptamers, we isolated DNA from bound and unbound fractions and quantified enrichment using PCR and qPCR with sex-specific primers. We observed significant differences in cycle threshold (Ct) values, indicating a greater proportion of X sperm in the unbound fraction than in the Y sperm with the Y aptamer, and vice versa for the X aptamer. In vitro fertilization (IVF) experiments further validated efficacy of these aptamers, with APT 2Y resulting in over 78 % enrichment of X sperm, whereas APT 1Y resulted in more than 65 % enrichment. This study advances aptamer-based methods for selective sperm enrichment by integrating SELEX-based aptamer selection, IVF embryo outcomes and PCR-based embryo sexing, offering promising prospects for sex-specific selection in livestock breeding.