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We established a short-term (24 h) culture system for bovine oviduct epithelial cells (BOECs), obtained on day 3.5 of the estrous cycle and evaluated the cells with respect to morphological criteria, marker gene expression, and hormone responsiveness. BOEC sheets were isolated mechanically from the ampulla with similar yields from oviducts ipsi- and contralateral to the ovulation site (57.9 ± 4.6 and 56.4 ± 8.0 × 10 ⁶ cells). BOECs showed > 95% purity and cells cultured for 24 h maintained morphological characteristics present in vivo , as determined by light microscopy, scanning electron microscopy, and transmission electron microscopy. Both secretory cells with numerous secretory granules and ciliated cells with long, well-developed, and vigorously beating kinocilia were visible. Quantitative real-time PCR failed to detect significant differences in transcript levels between ipsi …