Lectin Nanopurification Improves Post-Thaw Bovine Semen Quality

The spermatozoan glycocalyx mediates the initial contact with the female reproductive tract epithelia and oocyte vestments. This heterogeneous exocellular coating of glycans and glycosylated proteins can influence sperm maturation, motility, and fertilization potential. Targeting glycocalyx composition through nanopurification, a negative selection technique, can enrich bovine semen for specific sperm parameters. In this study, lectin-conjugated ferromagnetic nanoparticles (MNPs) targeting distinct surface glycans were evaluated in fresh bovine semen, individually and in combination. Lectin-conjugated MNPs were generated using Peanut agglutinin (PNA), Phaseolus lunatus (LBA), Lens culinaris agglutinin (LCA), and Ulex europaeus agglutinin I (UEA-I). Semen from twelve mature bulls (2-5 years old) was collected by electroejaculation, with two ejaculates pooled per collection. Sperm viability, acrosomal membrane integrity, mitochondrial membrane potential (MMP), and oxidative potential were analyzed by flow cytometry, while motility and morphology were assessed by computer-assisted semen analysis (CASA) and light microscopy before and after nanopurification as well as post-thaw. Nanopurification on fresh semen prior to cryopreservation using a combined lectin treatment (PNA, UEA-I and LBA; Mix1) increased MMP and UEA-I and Mix2 (UEA-I and LBA) nanopurification also decreased the prevalence of head morphology defects. Following cryopreservation, PNA, Mix1 and Mix2 improved acrosome membrane integrity, Mix2 increased the ability of the spermatozoa to adapt to oxidative stress and both PNA and Mix1 enhanced progressive motility compared to the no-sort control. Collectively, these findings demonstrate that lectin-based nanopurification preferentially removes functionally compromised spermatozoa and that combining lectins that target multiple glycans provides greater functional enrichment than most single lectin approaches.