RNA sequencing-based transcriptome profiling of dairy cows fed with a polyphenol rich grape pomace supplemented diet

The inclusion of some agro-industrial by-products in animals’ diet is becoming attractive not only for being part of an ambitious waste management and sustainability policies but also due to its possible nutritive values. The aim of this study was to evaluate the effect of grape pomace (GP) - the polyphenol-rich by-product of the wine industry – on the transcriptome of dairy cows. Twelve lactating Holstein-Friesian cows, homogeneous for age and lactation period, were assigned to two groups of six animals each - in a randomized pretest-posttest control group design. The first group received a basal diet and served as a control (CTR), while the other received a 10% GP-supplemented diet for 67 days. Whole blood was collected from each group at 2 time-points [beginning (T0), and after 67 days of the GP supplementation (Tf)], then total RNA was isolated, quality-controlled, and then used for library preparation. The sequencing of twenty-four samples (2 groups x 6 animals/group x 2 time-points) resulted in an average of 17 million reads per sample. The 50bp single-end reads were quality-controlled, mapped to the Bos taurus reference genome (UMD 3.1 assembly), then tested for the presence of differentially expressed genes (DEGs) in the same group (CTR or GP) after (compared with before) 67-days-supplementation period (Tf vs T0). On average, 95.6% of the reads were mapped to the reference genome. Reads mapped to exons were counted with HTSeq-count, then analysed by the DESeq2 R package. The bioinformatics analysis evidenced a significant (adjusted P < 0.1) change in expression of 14 and 88 genes in the CTR and GP groups, respectively. Four genes were found to be overlapping between the two groups, thus they were excluded from the GP group results as being ‘temporally’- and not experimentally-affected DEGs. Of the remaining 84 GP-affected genes, 73 were down-regulated, with most of them being ‘ribosomal protein’-coding genes. The functional analysis evidenced the positive enrichment of ‘defence response to other organism’ (P = 0.0002) biological process and the ‘interleukin signalling’ pathway (P = 0.0002), as well a negative enrichment of the ‘ribosome’ pathway (35 genes, P = 5x10-55). Overall, the transcriptomic signature of GP-supplemented diet reflects an induced immune system and a suppressed ‘ribosome biogenesis’, which can be ascribed to the GP’s polyphenol-derived antioxidant effect.