MicroRNA Sequencing Reveals the Effect of Different Levels of Non-Fibrous Carbohydrate/Neutral Detergent Fiber on Rumen Development in Calves

SIMPLE SUMMARY: By histological sectioning and staining of rumen tissues from calves fed with a high or low ratio of non-fibrous carbohydrate/neutral detergent fiber diet, we found that the length and width of papillae were significantly affected by the ratio. From microRNA expression analysis we found cell proliferation, differentiation, physical and nutrient stimuli processes participate in the development of the rumen. In addition, bta-miR-128 was found to affect rumen development by negatively regulating PPARG and SLC16A1. Our findings provided an important resource for the continuing study of rumen development and absorption. ABSTRACT: Rumen development in calves is affected by many factors, including dietary composition. MicroRNAs (miRNAs) are known to function in the development of the rumen in cattle, what is not known is how these miRNAs function in rumen development of calves fed with high and low ratios of non-fibrous carbohydrate (NFC)/neutral detergent fiber (NDF). A total of six healthy Charolais hybrids bull calves of similar weight were divided into two groups; three calves were fed a mixed diet with NFC/NDF = 1.35 (H group), and three were fed a mixed diet with NFC/NDF = 0.80 (L group). After 105 days on the diet, calves were sacrificed and rumen tissues were collected. Tissues were subjected to histological observation and miRNA expression analysis. Functional enrichment analysis was conducted on the target genes of the miRNAs. Targeting and regulatory relationships were verified by luciferase reporter assay and quantitative PCR (qPCR). We found that the length of rumen papilla in the L group was significantly greater than that in the H group, while the width of rumen papilla in H group was significantly greater than that that in L group. We identified 896 miRNAs; 540 known miRNAs, and 356 novel predicted miRNAs. After statistical testing, we identified 24 differentially expressed miRNAs (DEmiRNAs). miRNA-mRNA-cluster network analysis and literature reviews revealed that cell proliferation, differentiation, physical and nutrient stimuli processes participate in rumen development under different NFC/NDF levels. The regulatory relationships between three DEmiRNAs and five target genes were verified by examining the levels of expression. The binding sites on bta-miR-128 for the peroxisome proliferator activated receptor gamma (PPARG) and solute carrier family 16 member 1 (SLC16A1) genes were investigated using a dual luciferase assay. The results of this study provide insight into the role of miRNAs in rumen development in calves under different NFC/NDF levels.