Early fetal sex identification in the Piau pig breed using Y chromosome-linked transcripts

Sexual differences in embryos and fetuses are traditionally attributed to the development of the reproductive system and the production of sex hormones. In this study, the sex of Piau pig conceptuses (25-day embryos and 35-day fetuses) was determined using RNA-seq data through the detection of Y chromosome-linked transcripts. After quality control, 17 conceptuses were analyzed (8 embryos and 9 fetuses). Sex classification was based on the sum of counts per million (ΣCPM _chrY) of twelve candidate genes, with thresholds above 200 for males and below 2 for females, wich allowed consistent separation between sexes. Among the identified transcripts, five genes (LOC102162178, ENSSSCG00000025253, USP9Y, LOC110257938, and ENSSSCG00000047835) are reported for the first time in association with sex determination in pigs. Network analysis revealed regulatory associations between these transcripts and transcription factors involved in gonadal development, including SRY, SOX9, and NOBOX. This study demonstrates that RNA-seq enables sex identification at early stages of swine development and reveals novel Y chromosome-linked transcripts in the Piau breed and their potential regulatory associations during swine sexual differentiation.