# Integrated Analysis of Proteomics and Metabolomics for Heat Stress in Chinese Holstein Cows

## TL;DR

This study explores how Chinese Holstein cows respond to heat stress by analyzing proteins and metabolites, identifying key pathways and potential biomarkers for heat tolerance.

## Contribution

The study identifies novel protein-metabolite interactions and potential biomarkers for heat stress in Chinese Holstein cows.

## Key findings

- 29 differentially expressed proteins and 338 differential metabolites were identified in heat-stressed and heat-resistant cows.
- Four key pathways were revealed, involving up-regulated PLOD1 and ACTN4 and down-regulated EXT1 and GSN.
- Interference of the ACTN4 gene induces apoptosis in mammary epithelial cells, suggesting its role as a potential biomarker for heat stress.

## Abstract

Heat stress (HS) severely jeopardizes thermoregulation in dairy cows, significantly reducing productivity. The present study revealed 29 differentially expressed proteins and 338 differential metabolites by using TMT-based proteomes and an untargeted metabolomics approach in the heat-stressed (n = 6) and heat-resistant (n = 6) groups, respectively. Combined analysis revealed four key pathways underlying protein–metabolite interactions, where up-regulated PLOD1 and ACTN4 and down-regulated EXT1 and GSN interacting with the down-regulated N6-Acetyl-L-lysine, citric acid, 4-Pyridoxic acid, uracil, and uric acid and the up-regulated arachidonic acid were enriched. Interference of the ACTN4 gene could induce dairy cow mammary epithelial cells apoptosis, which could be regarded as a potential biomarker for HS in Chinese Holstein. These findings provide new insights into the molecular mechanisms underlying HS in Chinese Holstein.

Heat stress (HS) severely significantly reduces milk yield and causes substantial economic losses of dairy cows. TMT-based proteomes and an untargeted metabolomics approach were used to conduct the proteomics and metabolomics in heat-stressed (HS, n = 6) and heat-resistant (HR, n = 6) Chinese Holstein. The proteomics showed that 29 differentially expressed proteins (DEPs), with SERPINA3-7, ACTN4, and PLOD1 up-regulated, and GSN down-regulated in HR cows. The metabolomics showed that 168 differential positive metabolites and 170 differential negative metabolites were identified, with HR cows exhibiting lower levels of anti-inflammatory compounds, such as N6-Acetyl-L-lysine. In addition, 29 DEPs and 338 metabolites revealed four key pathways, including the lysine degradation (ko00310) and metabolic pathway (ko01100) with underlying protein–metabolite interactions, where up-regulated PLOD1 and ACTN4 and down-regulated EXT1 and GSN were observed to be interacting with the down-regulated N6-Acetyl-L-lysine, citric acid, 4-Pyridoxic acid, uracil, and uric acid, and the up-regulated arachidonic acid was enriched, which could be used for rapid and noninvasive screening of heat-tolerant cows. Functional validation through cell experiments, qPCR, and Western blot analyses showed that the interference of the ACTN4 gene could induce dairy cow mammary epithelial cell apoptosis, which could be regarded as a potential biomarker for HS in Chinese Holstein. Our results facilitate a better understanding of the molecular mechanism underlying the HS issue in dairy cows and provide a crucial insight into the alternative strategies to enhance animal welfare and productivity under high-temperature conditions.