Abstract
Heat stress (HS) is a major issue affecting productivity and physiological responses in dairy cattle. Advances in gene function research have made it possible to identify favorable genotypes for HS tolerance. This study evaluates the combined effects of temperature–humidity index (THI) and genomic load of favorable SNPs in the genes GRM8, SMAD3, and TLR4 on daily milk yield (DMY), rectal temperature (RT), and gene expression in Holstein cows (n = 160). Environmental conditions ranged from thermoneutral to severe HS, allowing the assessment of genotype by environment interactions. A subset of cows (n = 40) was selected based on genomic load to evaluate gene expression under opposite thermal conditions. Results showed that increasing THI was associated with reduced DMY and increased RT, with more pronounced effects in cows carrying fewer favorable genotypes. Slope analyses revealed that cows with higher genomic load exhibited a moderate decline in DMY and lower increase in RT, indicating enhanced resilience to HS. Significant THI × genomic load interactions were detected above a threshold of 79 units (p < 0.05). Gene expression analyses supported these findings, showing differential expressions of GRM8, SMAD3, and TLR4 under HS conditions. The functional and predictive value of these genes as markers of thermotolerance could help producers by supporting their application in genetic selection programs, thus providing another tool to combat HS and improve resilience and productivity in dairy systems.