Numerous prediction equations have been developed based on mid-infrared (MIR) spectra and some could be potentially used as biomarkers of heat stress. However, practical experience shows that confusion between the effect of heat stress and other effects like lactation stage or feeding variation over the year can easily occur. On this basis, the objective of this study was to identify potential milk components predicted by MIR as biomarkers of heat stress based on a 2-step approach allowing to correct for those effects. The first step consisted in the estimation of residuals from test-day random regression models on days in milk (DIM) to remove systematic lactation stage effects. These models contained also, among other, general (i.e., month of production) or specific (i.e., herd × test-day (HTD)) fixed effects related to feeding and management. During the second step, means and variances of residuals by temperature and humidity index (THI) classes were studied. The models were applied to 611,063 records from 97,042 primiparous Holstein cows from 2015 to 2022 in the south of Belgium. The MIR-predicted milk components with the highest deviations from the mean with increasing THI were protein percentage, casein concentration, magnesium (Mg) concentration and, to a lesser extent, polyunsaturated fatty acid (PUFA) concentration. Concerning residual variances, the highest heteroscedasticity with THI was obtained for milk MIR monounsaturated fatty acid (MUFA), C18:1 cis-9 and citrate concentrations. Conversely, a relative homoscedasticity of variance with increasing THI was observed for several milk MIR components including protein percentage and casein concentration. Based on the criteria of the "good biomarkers" guidelines, milk protein percentage seems to be the most promising trait of this study, followed by Mg concentration. However, in the context of genetic evaluation which requires variability, milk MIR MUFA, C18:1 cis-9 or citrate concentration variations, if they are heritable, could be of great interest. Finally, an increase in milk MIR citrate concentration variance could be an early warning for the detection of heat stress in the frame of dairy herd improvement (DHI).
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