Deep sequencing shows microRNA involvement in bovine mammary gland adaptation to diets supplemented with linseed oil or safflower oil

Abstract

Background: Bovine milk fat composition is responsive to dietary manipulation providing an avenue to modify the content of fatty acids and especially some specific unsaturated fatty acid (USFA) isomers of benefit to human health. MicroRNAs (miRNAs) regulate gene expression but their specific roles in bovine mammary gland lipogenesis are unclear. The objective of this study was to determine the expression pattern of miRNAs following mammary gland adaptation to dietary supplementation with 5 % linseed or safflower oil using next generation RNA-sequencing.

Methods: Twenty-four Canadian Holstein dairy cows (twelve per treatment) in mid lactation were fed a control diet (total mixed ration of corn:grass silages) for 28 days followed by a treatment period (control diet supplemented with 5 % linseed or safflower oil) of 28 days. Milk samples were collected weekly for fat and individual fatty acid determination. RNA from mammary gland biopsies harvested on day-14 (control period) and on days +7 and +28 (treatment period) from six randomly selected cows per treatment was subjected to small RNA sequencing.

Results: Milk fat percentage decreased significantly (P < 0.001) during treatment with the two diets as compared to the control period. The individual saturated fatty acids C4:0, C6:0, C8:0, C14:0 and C16:0 decreased significantly (P < 0.05) while five USFAs (C14:1, C18:1n11t, C20:3n3, C20:5n3 and CLA:t10c12) increased remarkably (P < 0.05) in response to both treatments. Analysis of 361 million sequence reads generated 321 known bovine miRNAs and 176 novel miRNAs. The expression of fourteen and twenty-two miRNAs was affected (P < 0.05) by linseed and safflower oil treatments, respectively. Seven miRNAs including six up-regulated (bta-miR-199c, miR-199a-3p, miR-98, miR-378, miR-148b and miR-21-5p) and one down-regulated (bta-miR-200a) were found to be regulated (P < 0.05) by both treatments, and thus considered core differentially expressed (DE) miRNAs. The gene targets of core DE miRNAs have functions related to gene expression and general cellular metabolism (P < 0.05) and are enriched in four pathways of lipid metabolism (3-phosphoinositide biosynthesis, 3-phosphoinositide degradation, D-myo-inisitol-5-phosphate metabolism and the superpathway of inositol phosphate compounds).

Conclusion: Our results suggest that DE miRNAs in this study might be important regulators of bovine mammary lipogenesis and metabolism. The novel miRNAs identified in this study will further enrich the bovine miRNome repertoire and contribute to understanding mammary gland biology.

Fig. 1

Response of test day milk fat percentage to 5 % linseed oil or 5 % safflower oil treatments (***P < 0.001; **P < 0.01, ns P > 0.05)

Fig. 2

Raw data filtering and mapping statistics. a The statistics of raw reads from the preprocessing and mapping steps. A total of 387.4 million raw reads were processed. A total of 11.9 million reads were filtered during the preprocessing step (aqua bar). Reads that could not map to the bovine genome were 115.3 million (purple bar). This number (115.3 million) includes reads which mapped to more than 5 genomic locations. Finally, reads that mapped successfully to the bovine genome (Bta_4.6.1) were 260.2 million (red bar). b The relative abundance of different classes of small RNAs in the total reads that were successfully mapped to the bovine genome; (c) Length distribution of the mapped reads across all the libraries

Fig. 3

Top10 highly expressed miRNAs in bovine mammary gland following supplemental feeding with 5 % linseed oil and 5 % safflower oil

Fig. 4

Distribution of known miRNAs (blue bar) and novel miRNAs (red bar) across bovine chromosomes (a) and bovine genomic regions (b)

Fig. 5

Results of qPCR validation of the expression of four miRNAs in linseed (a) and safflower (b) treatments, when comparing day+28 with day-14

Fig. 6

Functional annotation and enriched canonical pathways of core differentially expressed miRNA targets. a Functional annotation of all the targets by core differentially expressed miRNAs (b) Enriched canonical pathways related with lipid metabolism

Fig. 7

Two functions of core miRNAs with the highest number of targets related with lipid metabolism. Red color indicates up-regulated miRNAs; Green color indicates down-regulated miRNAs; Yellow color shows genes targeted by more than one miRNAs