Determining the timeline of gonadal and genital differentiation in male and female equine fetuses allows for early detection and intervention in malformations

Sexual differentiation in the equine fetus involves coordinated morphogenetic processes that shape both the gonads and the genital ducts. Although the formation of testes and ovaries has been relatively well documented, the temporal dynamics and morphometric patterns of the mesonephric (Wolffian) and paramesonephric (Müllerian) ducts remain insufficiently characterized. This study aimed to detail the timeline of gonadal and ductal differentiation in male and female equine fetuses during the critical developmental window between 60 and 80 days of gestation. Gross anatomical evaluation, histological analysis, and immunohistochemistry for 17β-hydroxysteroid dehydrogenase (17β-HSD) were performed to assess structural maturation and steroidogenic activity. The mesonephric duct was quantitatively assessed using unbiased stereological methods. Results demonstrated clear sexual dimorphism: males exhibited increased mesonephric duct volume at 80 days, whereas females showed progressive regression of this structure. Notably, this preliminary comparison across two ages appears to quantitatively support prior descriptions of paramesonephric duct regression. Positive 17β-HSD immunolabeling in both sexes indicated active steroidogenesis during this developmental window. By establishing a refined timeline for gonadal and ductal differentiation, this study advances the understanding of normal reproductive tract development and supports early identification of congenital malformations in horses.