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Accurate pathogen detection in bovine semen is crucial for animal health surveillance and international trade. Semen presents unique challenges due to the presence of PCR inhibitors from seminal plasma and extender components, reducing nucleic acid extraction efficiency and sensitivity. The two National Animal Health Laboratory Network-approved extraction platforms (MagMAX CORE and IndiMag Pathogen Kits) were evaluated using 88 negative extended semen samples at 200 µL input volume, reduced input volumes, and pretreatment strategies with two influenza A virus (IAV) PCR assays, containing different exogenous internal controls (ICs) to assess PCR inhibition. The ICs yielded overall passing rates from 31.8% to 100.0% and varied greatly based on the extender formulation and extraction protocol. Validation continued with naturally infected semen containing Mycoplasma bovis, bovine viral diarrhea virus, bovine herpesvirus-1, and limit of detection using Mycoplasma bovis before selecting the IndiMag Pathogen 100-na for evaluation of diagnostic sensitivity and specificity, reproducibility, and detection limits with IAV-spiked samples, using the two IAV PCR assays and their ICs. The naturally infected semen samples were screened and were negative for IAV by both PCR assays. These findings underscore the importance of tailored extraction methods in overcoming semen-associated inhibition and facilitating reliable pathogen surveillance in bovine germplasm.