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.

Background: Single nucleotide polymorphisms (SNPs) are widely used genetic markers with applications in human disease diagnostics, animal breeding, and evolutionary studies, but existing genotyping methods can be labor-intensive and costly. The aim of this study is to develop a simple and rapid method for identification of a single nucleotide change. Methods: A modified Polymerase Chain Reaction Amplification of Multiple Specific Alleles (PAMSA) and high resolution melt (HRM) analysis was performed to discriminate a bovine polymorphism in the NCAPG gene (rs109570900, 1326T > G). Results: The inclusion of tails in the primers enabled allele discrimination based on PCR product lengths, detected through agarose gel electrophoresis, successfully determining various genotypes, albeit with some time and labor intensity due to the use of relatively costly high-resolution agarose gels. Additionally, high-resolution melt (HRM) analysis with tailed primers effectively distinguished the GG genotype from the TT genotype in bovine muscle cell lines, offering a reliable way to distinguish SNP polymorphisms without the need for time-consuming AS-PCR. Conclusions: Our experiments demonstrated the importance of incorporating unique mismatched bases in the allele-specific primers to prevent cross-amplification by fragmented primers. This efficient and cost-effective method, as presented here, enables genotyping laboratories to analyze SNPs using standard real-time PCR.