First visualization of intact sperm storage tubules in the duck oviduct: A fluorescent labeling approach for targeted SST sampling.

Sperm storage tubules (SST) are central to fertility in birds, yet their structure and anatomical location have not been well characterized in ducks. This gap has limited direct study of the mechanisms that drive variation in fertility persistence among females used in intergeneric crosses. Here, we labeled duck spermatozoa with Hoechst 33342 and traced their distribution in the oviduct after artificial insemination. Staining conditions were optimized across temperature, dye concentration, and incubation duration. The selected protocol (40 µg/mL, 40 min, 37°C) produced strong fluorescence without compromising motility. Labeled spermatozoa were inseminated into Wujie White duck females, a line bred for prolonged fertility persistence in crosses with Muscovy drakes. Oviducts collected 24 hr after the second insemination were examined by fluorescence microscopy, revealing clusters of labeled spermatozoa within the mucosal folds of the utero-vaginal junction (UVJ). Hematoxylin and eosin staining confirmed that SST were present exclusively at the UVJ of the lower oviduct. Unlike conventional histology, the present approach combines fluorescent sperm labeling, artificial insemination, and direct examination of isolated UVJ villi. This permits concurrent evaluation of SST morphology and intraluminal sperm distribution at the individual level. It can also serve as a practical tool for future comparative studies of sperm retention in females with contrasting fertility persistence. Histological examination of five additional non-inseminated females further verified the consistent presence of SST in UVJ villi across individuals. To our knowledge, this study represents the first direct visualization and localization of intact SST in the duck oviduct, and defines an anatomical reference and targeted sampling strategy for reproductive research in waterfowl.