Insemination-driven epithelial remodeling and TLR4-mediated immune activation underlie sperm storage tubule maturation in young female kadaknath chickens

Abstract

Long-term sperm storage in the avian utero-vaginal junction (UVJ) is essential for reproductive efficiency, yet the developmental status of sperm storage tubules (SSTs) and their immunological regulation in young laying hens remains poorly understood. This study examined whether SSTs are functionally competent prior to insemination and how insemination influences their maturation in the indigenous Kadaknath chicken breed. Forty, six-month-old with no prior exposure to natural mating or artificial insemination hens were assigned to either non-inseminated controls or artificially inseminated groups. UVJ tissues were collected at 0, 1, 6, and 20 hours post-insemination and analyzed using histological and immunohistochemical techniques, with a focus on Toll-like receptor 4 (TLR4) expression. In non-inseminated hens, SSTs were sparse, structurally immature, and indicative of a non-functional state. Artificial insemination induced a highly organized, epithelial remodelling process involving invagination of ciliated mucosal folds, gradual loss of cilia, SST separation, lumen formation, and eventual establishment of mature, non-ciliated SSTs. Concomitantly, insemination led to a marked upregulation of TLR4 expression in the UVJ mucosa, whereas negligible expression was observed in non-inseminated hens. This insemination-dependent activation of innate immune signalling highlights a coordinated interaction between epithelial differentiation and immune responsiveness during SST maturation. Overall, the findings demonstrate that SSTs in young Kadaknath hens are developmentally primed but require insemination as a physiological trigger for structural and immunological maturation, offering valuable insights for fertility management and conservation of indigenous poultry breeds