| Abundant research has confirmed that dietary supplementation of plant oils(soybean oil,linseed oil, safflower oil and fish oil) which are rich in unsaturated fatty acids(USFA) could result in milk fat depression during which the de novo synthesis of short and medium fatty acids will be suppressed whereas the level of USFA was increased. Thus the milk fatty acid profile will be greatly altered and agree better with human needs. Furthermore, this milk fat depression also serves an ideal model for studying molecular mechanisms underlying milk fat synthesis and regulation.Milk fat synthesis relies on a complexed regulating network which involves many key lipogenic genes and transcription factors. Noncoding RNA(mi RNA and long noncoding RNA)has been shown to be important regulators of gene expression. Also studies have evidenced the involvement of noncoding RNA in fatty acid synthesis and muscle differentiation as well as involvement of miRNA in mammary gland development, milk synthesis and regulation of milk lactation cycle. However, it is still not clear whether noncoding RNA participated in milk fat synthesis. This study has selected 36 Canadian dairy cows in mid lactation to study the effects of dietary supplementation of 5% linseed oil or safflower oil on milk fat synthesis as well as on the miRNA and lncRNA transcriptome of mammary gland. We also used bioinformatics methods to study the biological functions of the noncoding RNA in milk fat synthesis. Furthermore, studies in recent years have observed abundant presence of RNA in milk. Thus we have studied and compared miRNA profiles in milk to determine whether milk could be an alternative method of mammary biopsy and the result of our work will also serve as a reference for further study of milk miRNA functions.1. Effect of dietary supplementation of 5% linseed oil or safflower oil on milk fat synthesis and fat compositionLinseed oil and safflower oil both caused a significant reduction of 34.2% and 29.9%respectively in milk fat percentage at Day+28 as compared to Day-14.The two treatments posed a similar effect on the yield of SFA, with 6 SFA(4:0, C6:0,C8:0, C14:0, C16:0 and C17:0) significantly reduced while yield of C18:0 was significantlyincreased at Day+7 and then returned to the level of control period. Four of the USFA(C18:1n11t, C20:3n3, C20:5n3 and CLA:10t12c) were significantly increased in response to the two treatments while C14:1 was significantly reduced at Day+28 as compared to Day-14.2. Effect of dietary supplementation of 5% linseed oil or safflower oil on mi RNome of mammary glandThis study has established a comprehensive workflow for miRNA-seq data analysis and analyzed miRNome of mammary gland. Twenty-seven miRNAs were found to be differentially expressed(DE) at the late treatment period(Day+28).In mammary gland, 321 known miRNA and 176 novel miRNA were expressed. The most ten highly expressed miRNA account for 70.48% of total reads counts of miRNA.Fourteen and twenty-two mi RNA were differentially expressed in response to linseed oil and safflower oil supplementation respectively. Seven of the DE mi RNA were shared by the two treatments and thus defined as core DE mi RNA. Function analysis using IPA suggested that the core miRNA were related with fatty acid synthesis through affecting concentration of lipid and synthesis of lipid.3. Effect of dietary supplementation of 5% linseed oil or safflower oil on lncRNome of mammary glandThis study has established a complete and reliable workflow for lncRNA analysis which allowed us to investigate characteristics of lncRNome and effect of dietary supplementation of 5% linseed oil or safflower oil on lncRNome of mammary gland.A total of 4113 lncRNA genes encoding 6497 transcripts were expressed in bovine mammary gland tissue.In linseed oil and safflower oil groups, 140 and 85 lncRNA were differentially expressed respectively. The DE lncRNA appeared at early treatment period(Day+7) and late treatment period(Day+28).4. Study of milk miRNome and comparison with those of mammary gland tissueThis study has established and improved RNA extraction methods from milk fractions(milk fat, whey and somatic cells) and has successfully constructed small RNA sequencing library for downstream analysis.A total of 210, 200 and 249 known miRNA were expressed in milk fat, whey and somatic cells respectively with 33, 31 and 36 novel miRNA discovered in corresponding fractions.Comparison of milk miRNome(milk fat, whey and somatic cells) with those of mammary gland showed that the miRNome of milk fat was most closely related with mammary gland tissue which suggested that milk fat could be used as a replacement ofmammary biopsy approach.Fourteen of the twenty highly expressed miRNA were shared among milk fat, whey and cells with specifically enriched miRNA present in each fraction. The fourteen highly expressed miRNA were related with milk synthesis as well as tissue and organ development. |
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