Characterization of microRNA in cow milk and colostrum

Abstract

MicroRNAs (miRNAs) are short RNA molecules, typically 21 to 25 nucleotides long, synthesized within eukaryotic cells. They play a crucial role in coordinating complex gene expression regulatory networks. The miRNAs are involved in post-transcriptional regulation, either by degrading target mRNA or suppressing their transcription, thereby influencing protein translation. Additionally, characteristics of farm production systems exert a noticeable influence on miRNA profiles, shaping the functional properties of bovine milk. In this study, milk and colostrum samples of Holstein cows (Bos taurus) were collected for RNA extraction and sequencing. Sequencing data underwent quality controls, and short noncoding RNA reads were analyzed to identify and compare miRNA profiles. The sequencing runs yielded 73,567,661 and 44,283,978 reads for milk and colostrum samples, respectively. Following trimming, 8,335,860 and 7,778,212 reads for milk and colostrum samples were retained. A total of 4.3% milk and 37.8% colostrum reads were identified as miRNAs. Overall, 157 miRNAs were common to milk and colostrum, 2 were unique to milk, and 90 were unique to colostrum. After applying a minimal cutoff of 100 reads per miRNA, 146 miRNAs were considered as expressed. In milk, bta-miR-101 was unique; in colostrum, bta-miR-18a, bta-miR-262-3p, bta-miR-130a, and bta-miR-224 were unique; and 141 miRNAs were common to colostrum and milk. Chromosome distribution of these miRNAs varied, with higher representation on autosome 19 and sexual chromosome X. Overall, colostrum contained a greater number of miRNAs, although specific miRNAs such as miR-11-988b, miR-140, and miR-146b were more abundant in milk. Conversely, miRNAs such as miR-7a-5p, let-7e, and miR-16a were more prevalent in colostrum. Validation through quantitative PCR confirmed the expression levels of selected miRNAs from RNA sequencing, establishing concordance between the methods. Analysis of gene regulation by specific miRNAs identified key miRNAs (e.g., miR-223, miR-181a, and miR-155) involved in regulating various genes. Distinct miRNA profiles in milk and colostrum suggest various functionalities, potentially influencing calf development and immune responses. The study unveils a comprehensive map of miRNAs in milk and colostrum and highlights their potential roles in regulating biological processes, as they play an essential role in calf development, disease resistance, and metabolism.

Keywords: Holstein; bulk milk; miRNA; pool.