Local Regulation of Milk Synthesis Capacity in the Mammary Gland of Lactating Dairy Cows

Abstract

Lactating dairy cows heavily rely on mammary gland functionality to maximize milk production. The number and activity of secretory mammary epithelial cells (MEC) plays a pivotal role in defining the synthesis potential of the gland. This dissertation aimed to investigate the effects of increased milking frequency (IMF), heat stress (HS), and cell heterogeneity as key contributors to the regulation of mammary gland milk synthesis capacity in lactating Holstein cows. The first study evaluated the implementation of IMF with 2x and 4x udder halves at early and mid-lactation for 21 and 20 d on milk yield (MY) and its association with changes in cistern and alveolar capacity. Results showed that udder halves milked 4x produced 2.27 kg more MY. Additionally, cows milked during early and mid-lactation had increased cistern capacity, while alveolar capacity remained unaffected. This suggests that increased cistern capacity may support MY enhancement through possible systemic responses caused by IMF. The second study examined the effects of 4 days of HS on mammary gland tissue structure, MEC number, and activity using a pair feeding model. Heat stress reduced MY of 4.3 kg/d. At the tissue level, HS decreased alveolar area and increased alveoli number and nucleated MEC per area. Gene expression analysis revealed unaffected activity-related targets but showed reduced phosphorylation of protein synthesis (pSTAT5) and cell survival (pS6K1) markers, as well as upregulation of an autophagosome-related protein (LC3 II). These findings indicate impaired pathways that could explain the reduction in MY after acute HS. The final study utilized single-cell RNA sequencing (scRNA-seq) to characterize the heterogeneity of epithelial and immune cell subpopulations in milk. Analysis revealed multiple subpopulations with distinct gene expression profiles, including different subtypes of mammary epithelial cells expressing representative marker genes (CSN3, CSN2, CSN1S1, CSN1S2, and LALBA) and immune cell types such as T cells, granulocytes (including neutrophils), macrophages, and B cells. Understanding the populations of hematopoietic cells in milk provides valuable insights into mammary gland function during lactation. The investigation of factors influencing cell number and activity in MEC is crucial for optimizing milk production and maintaining udder health. By identifying and addressing these factors, dairy farmers and researchers can implement strategies to enhance mammary gland function, improve milk production efficiency, and ensure the overall well-being of dairy cows.