A single-cell transcriptomic study of milk cells from dairy cows with divergent lactation performance.

High-lactation cows exhibit advantages in milk yield and quality compared to low-lactation cows; however, the underlying mechanisms remain unclear. Based on the demand for high-quality milk sources in the food industry, this study used single-cell sequencing technology (scRNA-seq) on the 10 × Chromium platform to analyze the milk cells of 10 Holstein cows (5 in the high-lactation group and 5 in the low-lactation group). The seven cell types included two types of epithelial cells (epithelial and secretory epithelial cells) and five types of immune cells (neutrophils, T cells, macrophages, B cells, and dendritic cells). Further sub-clustering analysis identified three epithelial cell types and nine T-cell subsets, and their differentiation paths were depicted through pseudo temporal analysis. Inter-group comparisons revealed differential genes and signaling pathways that affect lactation performance, such as lactation-related pathways (prolactin, protein export, thermogenesis) and immune-related pathways (Toll-like receptor, cytokine-receptor interaction, and NF-κB). In addition, this study elucidated the complex signaling relationships between epithelial and immune cells, especially the impact of CyPA, ICAM, and SELL signaling pathways on lactation. Moreover, additional analyses of macrophage and neutrophil subpopulations further revealed their interactions with epithelial cells, providing complementary insights into immune regulation during lactation.This study enriches the knowledge of cow lactation biology and provides a reference for the food industry to screen high-quality milk sources and optimize dairy processing technology.