Xiaotong Zhao, Hua Chang, Quanpeng Zhang, Zhuoyu Zhao, Sihui He, Zongyan Lu, Xun Xiang
Background/Objectives: Bovine heat-stress RNA-seq studies differ in tissue, age, physiological state, and exposure design. We asked which responses recur across these contexts and which depend on the evidence base. Methods: We reprocessed 107 libraries from five in vivo Bos taurus studies and synthesized within-study heat-minus-control log2 fold changes using restricted maximum-likelihood random-effects models with modified Knapp-Hartung inference. Eight tissue- and age-aware scenarios tested the cross-context estimate. A six-component heat-stress transcriptomic stability index (HSTSI) was benchmarked against five simpler rankings, and an independent mammary single-nucleus dataset provided cell-resolved comparison. Results: Of 266 FDR-significant pathways, 259 retained direction across all five study deletions and 21 remained significant in every deletion. Translation, ribosome, protein folding, endoplasmic-reticulum processing, proteasome, and heat-response programs formed the most recurrent axis. Four principal sensitivity scenarios retained 0.920-0.932 gene-direction agreement and 0.957-0.981 effect-rank correlation with the five-study analysis. In the single-nucleus dataset, leading-edge genes from the principal proteostasis programs showed 89.4-100% pooled-nucleus direction agreement and 94.7-100% agreement among published cluster-level differentially expressed genes. HSTSI had the highest mean top-200 held-out direction agreement (0.583 versus 0.516-0.562), with variation among folds. None of 16,756 genes met a modified Knapp-Hartung FDR below 0.10. IL1R2 and SDCBP2 were externally concordant, whereas GZMK and CD8A were context-dependent. Conclusions: A coordinated proteostasis program was the most transferable heat-stress signal. Energy remodeling, immune-associated bulk signals, and individual genes showed greater context dependence and define priorities for tissue-matched follow-up.