Summary: The last 2 months before birth and the first 2 months after birth represent a crucial window for organ development, including the mammary gland, in dairy cows. Traditionally, it was thought that allometric mammary growth began after weaning or around puberty. However, recent research indicates that significant early development of the mammary parenchyma occurs during the perinatal period and can influence a cow's future milk production. This early growth is highly sensitive to environmental and nutritional factors. Heat stress abatement and higher nutritional planes in early life increase mammary parenchyma growth and improve future productivity. Proper management during the perinatal period is essential to optimize lifetime milk yield.

Heat stress (HS) during the transition period disrupts metabolic adaptation and mammary gland (MG) remodeling. Methionine, a rate-limiting amino acid, supports cellular growth and stress responses through one-carbon metabolism pathways. We evaluated whether rumen-protected methionine (RPM) supplementation during the transition period preserves MG structure and function in heat-stressed multiparous Holstein cows. We hypothesized that RPM would mitigate the negative effects of HS on MG microstructure, gene expression, and cellular turnover. A total of 53 multiparous Holstein cows were enrolled at dry-off and assigned to diets with or without RPM (MET, CON). Four weeks prepartum, all MET and half of CON cows were fitted with electric heat blankets to induce HS (METHS, CONHS), while remaining CON cows were thermoneutral (CONTN). MG biopsies (8 and 28 days in milk; DIM) and milk samples were collected to assess microstructure, cellular turnover, gene expression, and mammary epithelial integrity. HS appeared to impair MG remodeling, with fewer but larger alveoli and a thicker epithelial layer, with a slight increase in epithelial cell proliferation, increased occludin fluorescence intensity, and increased milk concentration of K⁺. Meanwhile, RPM under HS increased stromal proliferation, lower occludin fluorescence intensity, and a reduced Na⁺:K⁺ ratio, indicating overall improved mammary epithelial integrity. Additionally, RPM partially promoted greater alveolar organization early postpartum and reduced lumen expansion, with coordinated changes in one-carbon metabolism, amino acid transport, and mTOR-related pathways. Overall, HS disrupts MG cellular dynamics, whereas close-up methionine supplementation partially improves mammary adaptability under chronic transition period HS.