Daily variation in milk HSP70 in dairy cows during a heat wave

Heat waves (HWs) are increasing in frequency and severity in many regions under climate change, posing challenges to the welfare and productivity of dairy cows. Physiological and environmental indicators are widely used to assess heat stress (HS) in dairy cows; however, the short-term temporal dynamics and lagged responses of molecular stress biomarkers during HW events under production conditions remain poorly characterized. This study aimed to evaluate daily variation in milk HSP70 during a HW and to assess its association with the environmental temperature-humidity index (THI) and sensor-derived reticulorumen temperature (RRT). The research was carried out over 5 consecutive days of HW in a pasture-based dairy farm in Camden, New South Wales, Australia, investigating 20 clinically healthy, third-parity Holstein Friesian cows. Daily milk samples were obtained during afternoon milking for HSP70 assessment using competitive ELISA. Environmental data were recorded at 1-min intervals to calculate THI, while RRT was continuously monitored at 10-min intervals using rumen bolus sensors. Associations between THI, RRT, and milk HSP70 were analyzed using repeated-measures correlations and univariable linear mixed models across lag windows of up to 50 h before sample collection, with adjustment for multiple testing across lag intervals. Exploratory classification performance was evaluated using receiver operating characteristic (ROC) analysis. The results revealed delayed associations between milk HSP70 and both THI and RRT. Associations between milk HSP70 and THI were strongest within an approximate delayed response window of 40-50 h after heat exposure, with repeated-measures correlations ranging from 0.57 to 0.84. Corresponding mixed-model analyses remained significant after adjustment for multiple testing using the Benjamini-Hochberg false discovery rate (FDR) procedure (FDR-adjusted P < 0.001), with R²_c values ranging from 43.79% to 75.66%. Similarly, associations with RRT were strongest within a delayed response window of approximately 48-50 h after heat exposure, with repeated-measures correlations ranging from 0.51 to 0.62. Corresponding mixed-model analyses remained significant (FDR-adjusted P < 0.001), with R²_c values ranging from 40.92% to 51.71%. In contrast, RRT responded rapidly to environmental heat load, showing the strongest associations with THI within the immediate response window of approximately 0-6 h (repeated-measures r = 0.57-0.67), while the corresponding mixed-model analyses remained significant (FDR-adjusted P < 0.001; R²_c = 48.15-57.21%). An exploratory milk HSP70 cut-point of approximately 550 ng/mL was identified, with a sensitivity of 0.782 and specificity of 0.909 for RRT-defined HS. Overall, the findings indicate that milk HSP70 exhibits a delayed response pattern following HW exposure, consistent with cumulative cellular stress, and may provide supplementary insights alongside environmental and physiological indicators for evaluating prolonged HS in dairy cows in pasture-based systems.