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In this study, we recorded production traits and collected mammary biopsies from cows under thermoneutral (TN), HS, and pair-fed (PF) conditions.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>Clinical measurements confirmed HS-induced physiological alterations. Compared with TN cows, HS cows exhibited reduced dry matter intake (DMI), milk yield, and yields of fat, protein, and lactose, along with increased water intake and milk urea nitrogen. The use of PF controls indicated that decreased DMI accounted for 45% of the milk-yield reduction, whereas direct HS effects accounted for the remaining reduction.<\/jats:p>\n                    <jats:p>\n                      We applied single-nucleus RNA-seq (snRNA-seq) on mammary biopsies to generate cell-resolved HS responses. We identified 14 distinct cell clusters, including epithelial, immune, and stromal populations. Under the TN condition, casein genes (e.g.,\n                      <jats:italic>CSN1S1<\/jats:italic>\n                      ,\n                      <jats:italic>CSN2<\/jats:italic>\n                      ) were broadly expressed across luminal cells but were attenuated under HS, whereas luminal alveolar cells showed relative upregulation. Heat shock protein genes were strongly induced by HS, primarily in epithelial clusters. Gene-set enrichment analyses revealed increased ribosomal activities across HS-responsive clusters and enrichment of protein folding and metabolic pathways in luminal alveolar cells, suggesting elevated proteostasis demands under stress.\n                    <\/jats:p>\n                    <jats:p>Pseudotime analysis positioned luminal cells along a progenitor-to-secretory trajectory under TN, accompanied by increased casein gene expression, whereas under HS, mature luminal cells shifted toward a homeostasis regulatory state. Cell\u2013cell communication analysis demonstrated HS-induced remodeling of interepithelial signaling, including altered ERBB4-mediated signaling from luminal hormone-sensing to alveolar lineages. Finally, transcription factor activity profiling highlighted cell type-specific HS-activated regulators and their downstream target genes.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>Together, this cell type-resolved atlas delineates how HS alters bovine mammary epithelial function, developmental state, and intercellular crosstalk. These findings point to proteostasis pressure, disrupted signaling pathways, and rewired regulatory networks as mechanistic contributors to reduced lactational performance under HS, offering insights for improving heat resilience in dairy cattle.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1186\/s40104-026-01468-x","type":"journal-article","created":{"date-parts":[[2026,7,16]],"date-time":"2026-07-16T09:04:43Z","timestamp":1784192683000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Single-nucleus RNA sequencing reveals cell type-specific responses to heat stress in bovine mammary gland"],"prefix":"10.1186","volume":"17","author":[{"given":"Xingtan","family":"Yu","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"family":"Shambhvi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Diego A.","family":"Ceballos","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marina M.","family":"Ferreira","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alejandra","family":"Zapata","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nirosh","family":"Seneviratne","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sabina","family":"Pokharel","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yifei","family":"Fang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guangsheng","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Francisco A.","family":"Leal Yepes","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joseph W.","family":"McFadden","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jingyue Ellie","family":"Duan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,7,16]]},"reference":[{"key":"1468_CR1","doi-asserted-by":"publisher","unstructured":"Key N, Sneeringer S, Marquardt D. 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