Background: South Korea has recently faced record-high temperatures, which have adversely affected dairy production. Holstein cows, the primary dairy breed globally, are particularly sensitive to heat stress. In contrast, Jersey cows have shown greater heat tolerance, as demonstrated by phenotypic studies. Methods: We investigated physiological and molecular responses to heat stress in Holstein and Jersey cows by measuring rectal temperature, milk yield, and average daily gain, confirming Holstein cows’ greater vulnerability. To explore molecular mechanisms, we analyzed circulating microRNA profiles from whole blood samples collected under heat stress and normal conditions using microRNA-sequencing. Differential expression patterns were compared between the two breeds to identify biological pathways associated with heat stress. Results: Four microRNAs (bta-miR-20b, bta-miR-1246, bta-miR-2284x, and bta-miR- 2284y) were significantly differentially expressed in both breeds under heat stress (|FC| ≥ 2, p < 0.05). Notably, bta-miR-20b and bta-miR-1246 were linked to corpus luteum function and progesterone biosynthesis, while bta-miR-2284x and bta-miR- 2284y were associated with immune responses. A comparison of 11 potential heat stress-related microRNAs identified in previous studies of Holstein cows revealed consistent expression trends in Jersey cows, albeit with lower fold changes, suggesting their superior heat resilience. Conclusions: Our study highlights the physiological and microRNA-based differences in heat stress responses between Holstein and Jersey cows. Jersey cows exhibited greater resilience, supported by more stable microRNA expression profiles and improved heat stress indicators, making them a promising breed for dairy production in increasingly hot climates.

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
    Experimental animals
    HS indicators
    Statistical analysis
    Blood collection
    MiRNA-sequencing experiment and statistical analysis
    Bioinformatics analysis
RESULTS
    Estimation of THI and blood collection
    Effects of HS on physiological changes
    Identification of DE miRNAs of HS cows
    Putative target gene and signalling pathway analysis
DISCUSSION
CONCLUSION
REFERENCES

• Jihwan Lee(Dairy Science Division, National Institute of Animal Science, Rural Development Administration, Cheonan 31000, Korea)
• Doosan Kim(Dairy Science Division, National Institute of Animal Science, Rural Development Administration, Cheonan 31000, Korea)
• Gyeonglim Ryu(Dairy Science Division, National Institute of Animal Science, Rural Development Administration, Cheonan 31000, Korea)
• Hyeonguk Baek(Dairy Science Division, National Institute of Animal Science, Rural Development Administration, Cheonan 31000, Korea)
• Sangbum Kim(Dairy Science Division, National Institute of Animal Science, Rural Development Administration, Cheonan 31000, Korea)
• Taejeong Choi(Dairy Science Division, National Institute of Animal Science, Rural Development Administration, Cheonan 31000, Korea) Corresponding author
• Byeonghwi Lim(Department of Animal Science and Technology, Chung-Ang University, Anseong 17546, Korea)
• Joohwan Kim(Dairy Biotechnology R&D Center, Seoul Milk Cooperation, Yangpyeong 12528, Korea)
• Seungmin Ha(Animal Genetic Resources Research Center, National Institute of Animal Science, Rural Development Administration, Hamyang 50000, Korea)
• Seunghwan Lee(Division of Animal and Dairy Science, College of Agriculture and Life Sciences, Chungnam National University, Daejeon 34134, Korea) Corresponding author
• Inchul Choi(Division of Animal and Dairy Science, College of Agriculture and Life Sciences, Chungnam National University, Daejeon 34134, Korea) Corresponding author