Association of plasma miRNAs with early life performance and aging in dairy cattle

Abstract

Early life performance traits in dairy cattle can have important influences on lifetime productivity. Poor health and fertility are of great economical and animal welfare concern. Circulating miRNAs have been linked to several livestock traits, including resistance to infection, fertility, and muscle development. This study aimed to identify circulating miRNAs associated with early life performance traits and aging in dairy cattle. Plasma samples from female calves (n = 12) identified retrospectively as differing in health, growth, and fertility outcomes prior to first calving were analyzed using PCR arrays detecting 378 miRNAs. Levels of 6 miRNAs differed significantly in calves with poor growth/fertility relative to controls (t-test: P<0.05). Additionally, general(ized) (non)linear mixed models identified 1 miRNA associated with average daily gain until weaning, 22 with live bodyweight at one year of age, 47 with age at first service, and 19 with number of infections before first calving. Out of 85 distinct miRNAs that were associated with at least one animal trait, 9 miRNAs were validated by RT-qPCR in a larger cohort (n = 91 animals), which included longitudinal plasma samples (calf, heifer, first lactation cow). Significant associations (P<0.05) involving individual miRNAs or ratios between miRNAs and early-life performance traits were identified, but did not retain significance after multiple testing adjustment. However, levels of 8 plasma miRNAs (miR-126-3p, miR-127, miR-142-5p, miR-154b, miR-27b, miR-30c-5p, miR-34a, miR-363) changed significantly with age, most prominently during the calf-to-heifer transition. Comparative RT-qPCR analyses of these miRNAs across 19 calf tissues showed that most were ubiquitously expressed. Online database mining identified several pathways involved in metabolism and cell signaling as putative biological targets of these miRNAs. These results suggest that miR-126-3p, miR-127, miR-142-5p, miR-154b, miR-27b, miR-30c-5p, miR-34a, miR-363 are involved in regulating growth and development from birth to first lactation (~2 years old) and could provide useful biomarkers of aging in cattle.

Fig 1. PCA plot generated using growth/fertility trait data.

Red axes show the direction of the trait (where AI denotes artificial insemination, Age at AI is age at first service, ADG-firstAI is average daily gain until first service, and ADG-weaning is average daily gain until weaning). Hierarchical clustering was used to identify three clusters, indicated by color; this produced a dendrogram used for selecting groups of calves for analysis. Group G included the worst performers from cluster 3, and Group C the best performers from cluster 1.

Fig 2. miRNA detection in calf plasma by PCR arrays: a) number of miRNAs detected at different Cq ranges in each sample and b) top 20 most abundant miRNAs (mean Cq +/- SE), after exclusion of spiked-in sequences.

Fig 3. Loess regression curve of miRNA levels against age (months).

Each miRNA was plotted separately and adjusted for technical variation (batch number). Except for miR-425-3p (FDR-adjusted P = 0.426), all other miRNAs were significantly associated with age (* GLMM: P<0.001; FDR-adjusted).

Fig 4. Estimated marginal means (+/- SE) derived from a model for the effect of age group on miRNA levels.

Significant pairwise comparisons between age groups are indicated above bars as stars (* P<0.05; FDR-adjusted).

Fig 5. Mean (+/- SE) levels of age-related miRNAs across body tissues in calves.

Stars indicate the overall effect of tissue type on miRNA expression (Wald tests of GLMM: * P<0.05; *** P<0.001; FDR-adjusted).