# Characterization of extracellular vesicles at parturition in dairy cows with late-gestation heat stress

## TL;DR

Heat stress in late pregnancy changes protein content in cow extracellular vesicles, affecting tissue repair and immune responses at birth.

## Contribution

Identified specific extracellular vesicle proteins altered by heat stress in cows, suggesting potential biomarkers for maternal recovery.

## Key findings

- Cooling cows increased extracellular matrix and coagulation proteins in EVs.
- Heat stress reduced immunoglobulin-related proteins in EVs.
- EV proteins may reflect improved recovery and lower immune stress with cooling.

## Abstract

Summary: This study investigated how late-gestation heat stress (HT) affects extracellular vesicle (EV) protein profiles in dairy cows at parturition. Plasma EV were isolated from heat-stressed and cooled (CL) cows and analyzed by mass spectrometry. A total of 684 proteins were identified, of which 20 differed significantly between treatments. The EV from cooled cows were enriched in extracellular matrix and coagulation proteins, including laminins, collagen IV, fibrinogen, and von Willebrand factor. The presence of these proteins in CL cows suggests that cooling enhances molecular pathways involved in tissue repair and postpartum recovery. Conversely, immunoglobulin-related proteins and specific receptors were reduced in cooled cows, potentially reflecting lower immune stress. These findings indicate that HT during late gestation alters EV-mediated signaling related to metabolism, immune modulation, and tissue remodeling at parturition. The identified proteins may serve as potential biomarkers for assessing maternal adaptation and recovery, emphasizing the importance of environmental cooling during the dry period. NTA = nanoparticle tracking analysis; trt = treatment.

Summary: This study investigated how late-gestation heat stress (HT) affects extracellular vesicle (EV) protein profiles in dairy cows at parturition. Plasma EV were isolated from heat-stressed and cooled (CL) cows and analyzed by mass spectrometry. A total of 684 proteins were identified, of which 20 differed significantly between treatments. The EV from cooled cows were enriched in extracellular matrix and coagulation proteins, including laminins, collagen IV, fibrinogen, and von Willebrand factor. The presence of these proteins in CL cows suggests that cooling enhances molecular pathways involved in tissue repair and postpartum recovery. Conversely, immunoglobulin-related proteins and specific receptors were reduced in cooled cows, potentially reflecting lower immune stress. These findings indicate that HT during late gestation alters EV-mediated signaling related to metabolism, immune modulation, and tissue remodeling at parturition. The identified proteins may serve as potential biomarkers for assessing maternal adaptation and recovery, emphasizing the importance of environmental cooling during the dry period. NTA = nanoparticle tracking analysis; trt = treatment.

•Heat stress during late gestation significantly alters EV content.•Cooling increased extracellular matrix and coagulation proteins, perhaps for tissue repair and postpartum recovery.•Cooling decreased immunoglobulin-related proteins and certain receptors in EV.•Changes in EV proteins may reflect improved recovery and lower immune stress with cooling.

Heat stress during late gestation significantly alters EV content.

Cooling increased extracellular matrix and coagulation proteins, perhaps for tissue repair and postpartum recovery.

Cooling decreased immunoglobulin-related proteins and certain receptors in EV.

Changes in EV proteins may reflect improved recovery and lower immune stress with cooling.

Extracellular vesicles (EV) are recognized as important mediators of cell-cell communication, capable of transferring various cargo between cells. Thus, EV are potential biomarkers of health status in heat-stressed cows. We hypothesized that heat stress during late gestation would alter the protein profile of EV at parturition in dairy cows. To test this, maternal plasma samples were collected within 2 h after parturition (n = 16–17/treatment) from cows exposed to late-gestation heat stress (HT) or an active cooling system (CL). The EV were isolated from plasma via single-step size exclusion chromatography. Preparations were checked for quality by transmission electronic microscopy and then quantified by nanoparticle tracking analysis before proteomic analysis. High-resolution MS was performed, and a two-sided, unpaired Welch's t-test was done on proteins showing at least 3 valid values in one group and at least 70% of valid values in the other group using log2 (label-free quantification intensity). The significance threshold was P ≤ 0.059, and proteins with a log2 fold change (CL/HT) above 1.2 or below 0.66 were considered significantly affected. Potential EV biomarkers from bovine plasma were detected, including several proteins significantly affected when comparing CL and HT. We noted more abundance in the CL group of laminin subunit gamma 1, α 2, and β 1; transferrin receptor protein 1; collagen IV; fibrinogen α chain, gamma-B chain, and β chain; von Willebrand factor; and SPN protein. Furthermore, in the CL group, the less abundant proteins included immunoglobulin domain lambda and heavy chains; HGF activator; protein HP-20 homolog; trafficking from endoplasmic reticulum to Golgi regulator (TFG) protein; amine oxidase 3; and collectin member 10. These findings suggest that these EV may play a role in modulating maternal metabolism and immune adaptations at the time of parturition within the maternal circulation. In addition, late-gestation heat stress alters the secretion of specific proteins that are involved in adaptations related to parturition. Further investigation is needed to better understand the functionality of EV during late gestation and the impact of heat stress on the molecules they carry.