Disease & heat resistance

Metabolomic pathways associated with thermal tolerance and susceptibility in Spanish Holstein cows

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Abstract

Heat stress is becoming an increasingly relevant challenge for dairy production. Individual variation in heat responsiveness has been repeatedly observed; however, the metabolic basis underlying this variability remains poorly explored. In this context, this study analyzes the metabolomic profiles from cows exhibiting differences in physiological disturbance signs under heat stress with the aim of searching for bio-indicators to be used for breeding purposes.Blood samples from 81 Holstein cows were collected in two herds located in Castile-La Mancha (Spain) during summer heat waves. Respiratory rates (RR) were recorded on resting animals between 15:00 h and pre-milking, avoiding periods immediately following feeding, to minimize variability. Animals were classified into two groups based on their RR, calculated as deviation from the group mean within the same housing area: heat-susceptible (SUS) cows, showing elevated respiratory rates and visible thermal discomfort, and heat-tolerant (TOL) cows, maintaining low respiratory rates and minimal signs of heat stress. Metabolomic data were generated using Metabolon's untargeted mass spectrometry platform, which detects a wide range of small molecules across multiple chromatographic and ionization modes. Raw peak areas were normalized by Metabolon using batch-median scaling and inter-batch alignment. Xenobiotic compounds and metabolites with outlier values were removed. Statistical analysis followed a two-step approach. First, a generalized linear model was applied to remove confounding effects; metabolites significantly influenced by herd or lactation stage (FDR ≤ 0.05) were statistically corrected, retaining only the standardized residuals for further analysis. Second, a partial least squares discriminant analysis (PLS-DA) was performed on these corrected data to handle high dimensionality and multicollinearity, maximizing the separation between SUS and TOL groups. Metabolites with Variable Importance in Projection (VIP) > 1 were selected as the most informative for pathway enrichment analysis in MetaboAnalyst 6.0.PLS-DA showed a partial but consistent separation between SUS and TOL cows. In SUS cows, the metabolites with the highest VIP values were mainly amino acids and phospholipids, suggesting increased protein catabolism, nitrogen turnover, and membrane remodeling under heat stress. In contrast, TOL cows showed higher levels of complex lipids, fatty acid-related metabolites, and antioxidant molecules, consistent with more efficient lipid use and protection against oxidative damage. Pathway analysis supported these patterns, highlighting phospholipid-related routes in SUS cows and beta-oxidation and glutathione-related pathways in TOL cows. These findings do not establish causal mechanisms, but they reveal consistent metabolic patterns that may serve as useful biomarkers to be used as auxiliary traits to select cows with a better ability to cope with heat.Acknowledgments: This work was supported by the Re-Livestock project (EU Horizon Europe, Grant Agreement No. 101059609). The Castile-La Mancha Friesian Association provided its support for all the experimental work.

Keywords: 2026

How to Cite:

Carabaà±o, M., Dà­az, C., González, C., Hernández-Pumar, A., López-Martà­nez, J., Meneses, C., Ramón, M. & Reinoso-Peláez, E., (2026) “Metabolomic pathways associated with thermal tolerance and susceptibility in Spanish Holstein cows”, World Congress on Genetics Applied to Livestock Production Digital Archive 2026(1): 2285489. doi: https://doi.org/10.31274/wcgalp.23723

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