Effect of dietary supplementation with linseed oil on the miRnome profile of the bovine mammary gland

Abstract Text: Linseed is particularly rich in alpha-linolenic acid (C18:3n3) and its supplementation in diets induces an increase in milk unsaturated fatty acid content (along with decrease in milk saturated fatty acids) with potential milk fat depression. The specific role of microRNAs (miRNAs), important post-transcriptional regulators of gene expression, in this dietary adaption remains unknown. Using next generation sequencing, the miRnome of the bovine mammary gland in response to dietary supplementation with 5% linseed oil on dry matter bases was studied. Thirteen high producing Holstein dairy cows in mid lactation were fed a control ration (total mixed ration of corn and grass silages) for 28 days followed by a treatment period (control diet supplemented with 5% linseed oil) of 28 days. Milk component yields including fat (%) were measured on a weekly basis. Mammary biopsies on three cows were performed on day-14 (control period) and on days +7 and +28 (treatment period). Results show a significant decrease (P<0.0001) in milk fat yield(%) during the treatment period (3.02±0.17) as compared to the control period (3.62±0.17). Nine libraries were constructed and subjected to 50bp smallRNA sequencing. A total of 103,796,305 raw reads were obtained and 93,218,009 retained after adaptor trimming and quality filtering. Of these, 69,210,197 (74.25%) were mapped to the bovine genome. A total of 338 known miRNAs (82.6% of mapped reads) were identified with more than 1 count per million in at least 6 libraries. Furthermore, 223 novel hairpins encoding for 212 novel miRNAs were identified. Five miRNAs (bta-miR-148a, miR-143, miR-26a, miR-30a-5p and miR-10b) were most highly expressed, accounting for 54.93% of reads of identified known miRNAs. As compared to day-14 (control period), ten miRNAs were significantly regulated (five up-regulated: bta-miR-4286, mir-199c, miR-199a-3p, miR-98 and miR-23b-3p; five down-regulated: bta-miR-484, miR-96, miR-200a, miR-335 and miR-2299-5p) at day+28 (P<0.01) while no significant regulation was detected at day+7 (P>0.01). About 5541 genes were predicted to be targeted by differentially expressed miRNAs. Function enrichment analysis showed significant enrichment of target genes for functions related with lipid metabolism (P<0.01).  In conclusion, our study revealed that several miRNAs were differentially expressed during the milk fat depression introduced by linseed oil supplementation, suggesting that these miRNAs could be important regulators of mammary lipid synthesis. Furthermore, novel miRNAs identified in this study will greatly enrich the bovine miRnome repertoire and also act as targets for further study of bovine mammary gland biology.

Keywords: microRNA, linseed oil, bovine mammary gland