Heat stress (HS) poses significant challenges to the dairy industry, resulting in reduced milk production, impaired reproductive performance, and compromised animal welfare. Therefore, understanding the molecular mechanisms underlying cellular responses to HS is crucial for developing effective strategies to mitigate its adverse effects. Heat shock protein 70 (HSP70) has emerged as a potential player involved in cellular thermotolerance in dairy cows. This review provides a comprehensive overview of the role of HSP70 as a molecular chaperone in cellular thermotolerance in dairy cows under HS. HSP70 facilitates proper protein folding and prevents the aggregation of denatured proteins. By binding to misfolded proteins, it helps maintain protein homeostasis and prevents the accumulation of damaged proteins during HS. Additionally, HSP70 interacts with various regulatory proteins and signaling pathways, contributing to the cellular adaptive response to HS. The upregulation of HSP70 expression in response to HS is regulated by a complex network involving heat-shock factors (HSFs), heat-shock element-binding proteins, and HSF co-chaperones. Therefore, HSP70 holds the potential to be a useful indicator of tissue stress due to its role in maintaining cellular balance, and as it is released both inside and outside cells in response to stress. Traditional methods of measuring HSP70 in blood samples are labor-intensive, and with the process being potentially stressful for the animals and may subsequently affect the results. Therefore, measuring HSP expression in cow's milk has shown promise as an easy, non-invasive, and accurate way to detect HS in dairy cows. Monitoring HSP70 levels in milk can be applied as a supplementary approach to identify HS or HS resistance of individual cows, selection of suitable animals and to guide targeted management strategies. However, despite the potential advantages of using HSP70 as a biomarker for monitoring HS on dairy cows, challenges remain in standardizing measurement protocols, establishing species-specific reference ranges, addressing inter-individual variations, and determining the specificity of changes in HSP70 due to HS. Future research should focus on developing non-invasive techniques for HSP70 detection, with consideration of climatic conditions, and unravelling the molecular interactions and regulatory networks involving HSP70.

Heat stress (HS) negatively affects the health, reproduction, and milk production of dairy cows, indicating a major challenge to animal welfare and farm profitability. Although environmental and physiological indicators are commonly used to monitor HS, cellular biomarkers such as heat shock protein 70 (HSP70) offer a promising approach for detecting HS at the molecular level. This research aimed to evaluate and compare HSP70 levels in the milk, blood, and saliva of lactating dairy cows across seasons, assess their environmental responsiveness using the temperature-humidity index (THI) as the standard environmental indicator of HS under temperate Australian conditions, and investigate the potential of milk HSP70 as a noninvasive biomarker of HS at the cellular level. Twenty multiparous Holstein Friesian cows were monitored across 4 seasons (summer, autumn, winter, and spring) over 1 yr, and HSP70 concentrations were measured using a competitive ELISA system. Weather data, including THI, as well as milk yield and composition traits, were also recorded. Results showed significant seasonal variation in HSP70 levels, with the highest concentrations observed in summer across all sample types. Blood exhibited the highest (416.45 ng/mL) HSP70 concentrations overall, followed by saliva (304.89 ng/mL) and milk (279.02 ng/mL). Milk HSP70 showed the highest variability (CV = 53.76%), indicating that additional factors beyond HS may influence its expression, whereas blood and saliva appeared to be more consistent. The associations between HSP70 concentrations in biological fluids were initially evaluated using Pearson correlation analysis. To account for repeated measurements in cows and identify key predictors, linear mixed-effects models with cow identification as a random effect were then fitted. Moderate positive correlations were found between the concentration of milk HSP70 and both blood (r = 0.58) and saliva HSP70 (r = 0.53), indicating that milk HSP70 may reflect systemic stress responses in dairy cows. Multivariable linear mixed models identified average THI as a significant predictor of HSP70 concentrations across fluids. In the milk HSP70 model, DIM was also a significant predictor, and milk yield was not. Overall, this study provides preliminary evidence that milk HSP70 may have potential as a noninvasive indicator of HS in dairy cows. These findings also contribute to a better understanding of HS physiology and may inform future approaches for herd-level monitoring and management under climate change.

Summary: This study evaluated the impact of milk sample handling on the detection of heat shock protein 70 (HSP70) in bovine milk by using a competitive enzyme-linked immunosorbent assay (ELISA) system. Samples were processed as skimmed before storage (SBS) or skimmed after storage (SAS) and stored under various conditions: room temperature (~22°C; T1), refrigeration (4°C; T2), freezing (-20°C; T3), and refrigeration with preservative (4°C + bronopol; T4). Samples were analyzed every 2 days over 15 days to assess HSP70 stability. Results showed that SBS samples stored at 4°C or -20°C for up to 3 days preserved HSP70 levels with minimal degradation (4.4% and 7.9%, respectively), whereas SAS samples showed significantly higher degradation (44.2%-53.9%). Room temperature storage led to the greatest degradation, and bronopol use did not consistently maintain HSP70 levels. These findings highlight the importance of optimizing milk storage to preserve HSP70, a potential biomarker for cellular and heat stress in dairy cows.

Heat shock protein 70 (HSP70) has emerged as a potential physiological indicator of heat stress (HS), recognized for its substantial upregulation in response to increased temperatures and cellular stress. The accurate measurement of HSP70 is crucial for its application in research and on-farm monitoring. Commercial ELISA kits are widely available and exhibit high sensitivity; however, their limited working ranges require the dilution of high-concentration samples, which can potentially introduce variability. A recently developed in-house competitive ELISA provides a cost-effective alternative, previously validated for bovine HSP70 detection; however, its performance across various biological matrices has not been systematically evaluated against commercially available systems. A secondary objective was to determine whether HSP70 levels measured in less-invasive matrices such as milk and saliva, correspond to those obtained from blood. This study compared an in-house competitive ELISA to a commercial sandwich ELISA kit for detecting HSP70 in milk, blood, and saliva samples of dairy cows. Samples were analyzed with optimized methods, and assay performance was evaluated using descriptive statistics, Pearson correlations, and linear regression models. As expected for assays using different formats and calibration systems, the in-house ELISA indicated elevated absolute concentrations across all matrices; however, these differences were systematic and did not affect the strong agreement between methods. Blood samples had the lowest CV in both assays (2.76% in-house; 5.89% commercial), indicating greater repeatability than milk (5.69% and 10.24%) and saliva (7.5% and 9.95%). The consistently lower CV for the in-house method across all fluids indicates that it is more precise and can lead to higher statistical power than commercial assays for a given sample size. Strong positive correlations were observed between assays across all matrices (milk r = 0.91; blood r = 0.88; saliva r = 0.87). Regression analysis validated these relationships, with R² values of 0.777, 0.833, and 0.752 for milk, blood, and saliva, respectively. The findings indicate that the in-house ELISA closely aligns with the performance of the commercial kit. Despite the lower detectable minimum concentration of the commercial assay, the in-house assay provides a more economical alternative for evaluating physiologically high and moderate HSP70 levels; however, the commercial kit remains widely available for purchase. Among biological matrices, plasma exhibited the highest cross-assay correlation, whereas milk and saliva showed consistent relative trends, indicating their potential as noninvasive alternatives for HS monitoring in dairy cows.