Optimizing Nucleic Acid Extraction from Extended Bovine Semen for Endemic and High-Consequence Pathogens

Accurate pathogen detection in bovine semen is crucial for animal health surveillance and international trade, particularly in light of the recent detection of highly pathogenic avian influenza H5 viruses in cattle. However, semen presents unique challenges due to the presence of PCR inhibitors from seminal plasma and extender components, which reduce nucleic acid extraction efficiency, resulting in decreased sensitivity and false negatives. The two National Animal Health Laboratory Network-approved extraction platforms, MagMAX CORE and IndiMag Pathogen Kits, were evaluated using 88 negative extended semen samples with two influenza A virus (IAV) PCR assays. Using standard 200 µL inputs, the two internal positive controls yielded overall passing rates, from 31.8% to 97.7%, and varied greatly based on the extender formulation. Modified protocols incorporating reduced input volumes and pretreatment strategies significantly improved recovery, with the IndiMag Pathogen 100-na and MagMAX CORE 12.5-pretreatment protocols both achieving 100% overall passing rates of the internal positive controls. Validation with naturally infected semen containing Mycoplasma bovis, bovine viral diarrhea virus, bovine herpesvirus-1, and with IAV-spiked samples further confirmed enhanced sensitivity, reproducibility, and detection limits. These findings underscore the importance of tailored extraction methods in overcoming semen-associated inhibition and facilitating reliable pathogen surveillance in bovine germplasm.