Pier-Luc Dudemaine

Staphylococcus chromogenes (SC) is a common coagulase-negative staphylococcus described as an emerging mastitis pathogen and commonly found in dairy farms. This study investigated the potential involvement of DNA methylation in subclinical mastitis caused by SC. The whole-genome DNA methylation patterns and transcriptome profiles of milk somatic cells from four cows with naturally occurring SC subclinical mastitis (SCM) and four healthy cows were characterized by next-generation sequencing, bioinformatics, and integration analyses. Comparisons revealed abundant DNA methylation changes related to SCM, including differentially methylated cytosine sites (DMCs, n = 2,163,976), regions (DMRs, n = 58,965), and methylation haplotype blocks (dMHBs, n = 53,098). Integration of methylome and transcriptome data indicated a negative global association between DNA methylation at regulatory regions (promoters, first exons, and first introns) and gene expression. A total of 1486 genes with significant changes in the methylation levels of their regulatory regions and corresponding gene expression showed significant enrichment in biological processes and pathways related to immune functions. Sixteen dMHBs were identified as candidate discriminant signatures, and validation of two signatures in more samples further revealed the association of dMHBs with mammary gland health and production. This study demonstrated abundant DNA methylation changes with possible involvement in regulating host responses and potential as biomarkers for SCM.

Background: Mastitis caused by different pathogens includingStreptococcus uberis(S. uberis) is responsible for huge economic losses to the dairy industry. In order to investigate the potential genetic and epigenetic regulatory mechanisms of subclinical mastitis due toS. uberis, the DNA methylome (whole genome DNA methylation sequencing) and transcriptome (RNA sequencing) of milk somatic cells from cows with naturally occurringS. uberissubclinical mastitis and healthy control cows (n = 3/group) were studied.Results: Globally, the DNA methylation levels of CpG sites were low in the promoters and first exons but high in inner exons and introns. The DNA methylation levels at the promoter, first exon and first intron regions were negatively correlated with the expression level of genes at a whole-genome-wide scale. In general, DNA methylation level was lower inS. uberis-positive group (SUG) than in the control group (CTG). A total of 174,342 differentially methylated cytosines (DMCs) (FDR < 0.05) were identified between SUG and CTG, including 132,237, 7412 and 34,693 DMCs in the context of CpG, CHG and CHH (H = A or T or C), respectively. Besides, 101,612 methylation haplotype blocks (MHBs) were identified, including 451 MHBs that were significantly different (dMHB) between the two groups. A total of 2130 differentially expressed (DE) genes (1378 with up-regulated and 752 with down-regulated expression) were found in SUG. Integration of methylome and transcriptome data with MethGET program revealed 1623 genes with significant changes in their methylation levels and/or gene expression changes (MetGDE genes, MethGETP-value < 0.001). Functional enrichment of genes harboring ≥ 15 DMCs, DE genes and MetGDE genes suggest significant involvement of DNA methylation changes in the regulation of the host immune response toS. uberisinfection, especially cytokine activities. Furthermore, discriminant correlation analysis with DIABLO method identified 26 candidate biomarkers, including 6 DE genes, 15 CpG-DMCs and 5 dMHBs that discriminated between SUG and CTG.Conclusion: The integration of methylome and transcriptome of milk somatic cells suggests the possible involvement of DNA methylation changes in the regulation of the host immune response to subclinical mastitis due toS. uberis. The presented genetic and epigenetic biomarkers could contribute to the design of management strategies of subclinical mastitis and breeding for mastitis resistance.

Milk fat and protein contents are among key elements of milk quality, and they are attracting more attention in response to consumers′ demand for high-quality dairy products. To investigate the potential regulatory roles of DNA methylation underlying milk component yield, whole genome bisulfite sequencing was employed to profile the global DNA methylation patterns of mammary gland tissues from 17 Canada Holstein cows with various milk fat and protein contents. A total of 706, 2420 and 1645 differentially methylated CpG sites (DMCs) were found between high vs. low milk fat (HMF vs. LMF), high vs. low milk protein (HMP vs. LMP), and high vs. low milk fat and protein (HMFP vs. LMFP) groups, respectively (q value < 0.1). Twenty-seven, 56 and 67 genes harboring DMCs in gene regions (denoted DMC genes) were identified for HMF vs. LMF, HMP vs. LMP and HMFP vs. LMFP, respectively. DMC genes from HMP vs. LMP and HMFP vs. LMFP comparisons were significantly overrepresented in GO terms related to aerobic electron transport chain and/or mitochondrial ATP (adenosine triphosphate) synthesis coupled electron transport. A total of 83 (HMF vs. LMF), 708 (HMP vs. LMP) and 408 (HMFP vs. LMFP) DMCs were co-located with 87, 147 and 158 quantitative trait loci (QTL) for milk component and yield traits, respectively. In conclusion, the identified methylation changes are potentially involved in the regulation of milk fat and protein yields, as well as the variation in reported co-located QTLs.

This study aimed to investigate the potential regulatory roles of miRNAs in calf ileum developmental transition from the pre- to the post-weaning period. For this purpose, ileum tissues were collected from eight calves at the pre-weaning period and another eight calves at the post-weaning period and miRNA expression characterized by miRNA sequencing, followed by functional analyses. A total of 388 miRNAs, including 81 novel miRNAs, were identified. A total of 220 miRNAs were differentially expressed (DE) between the two periods. The potential functions of DE miRNAs in ileum development were supported by significant enrichment of their target genes in gene ontology terms related to metabolic processes and transcription factor activities or pathways related to metabolism (peroxisomes), vitamin digestion and absorption, lipid and protein metabolism, as well as intracellular signaling. Integration of DE miRNAs and DE mRNAs revealed several DE miRNA-mRNA pairs with crucial roles in ileum development (bta-miR-374a—FBXO18, bta-miR-374a—GTPBP3, bta-miR-374a—GNB2) and immune function (bta-miR-15b—IKBKB). This is the first integrated miRNA-mRNA analysis exploring the potential roles of miRNAs in calf ileum growth and development during early life.

A better understanding of the factors that regulate growth and immune response of the gastrointestinal tract (GIT) of calves will promote informed management practices in calf rearing. This study aimed to explore genomics (messenger RNA (mRNA)) and epigenomics (long non-coding RNA (lncRNA)) mechanisms regulating the development of the rumen and ileum in calves. Thirty-two calves (≈5-days-old) were reared for 96 days following standard procedures. Sixteen calves were humanely euthanized on experiment day 33 (D33) (pre-weaning) and another 16 on D96 (post-weaning) for collection of ileum and rumen tissues. RNA from tissues was subjected to next generation sequencing and 3310 and 4217 mRNAs were differentially expressed (DE) between D33 and D96 in ileum and rumen tissues, respectively. Gene ontology and pathways enrichment of DE genes confirmed their roles in developmental processes, immunity and lipid metabolism. A total of 1568 (63 known and 1505 novel) and 4243 (88 known and 4155 novel) lncRNAs were detected in ileum and rumen tissues, respectively. Cis target gene analysis identified BMPR1A, an important gene for a GIT disease (juvenile polyposis syndrome) in humans, as a candidate cis target gene for lncRNAs in both tissues. LncRNA cis target gene enrichment suggested that lncRNAs might regulate growth and development in both tissues as well as posttranscriptional gene silencing by RNA or microRNA processing in rumen, or disease resistance mechanisms in ileum. This study provides a catalog of bovine lncRNAs and set a baseline for exploring their functions in calf GIT development.

Co-expression network analyses provide insights into the molecular interactions underlying complex traits and diseases. In this study, co-expression network analysis was performed to detect expression patterns (modules or clusters) of microRNAs (miRNAs) during lactation, and to identify miRNA regulatory mechanisms for milk yield and component traits (fat, protein, somatic cell count (SCC), lactose, and milk urea nitrogen (MUN)) via miRNA target gene enrichment analysis. miRNA expression (713 miRNAs), and milk yield and components (Fat%, Protein%, lactose, SCC, MUN) data of nine cows at each of six different time points (day 30 (D30), D70, D130, D170, D230 and D290) of an entire lactation curve were used. Four modules or clusters (GREEN, BLUE, RED and TURQUOISE) of miRNAs were identified as important for milk yield and component traits. The GREEN and BLUE modules were significantly correlated (|r| > 0.5) with milk yield and lactose, respectively. The RED and TURQUOISE modules were significantly correlated (|r| > 0.5) with both SCC and lactose. In the GREEN module, three abundantly expressed miRNAs (miR-148a, miR-186 and miR-200a) were most significantly correlated to milk yield, and are probably the most important miRNAs for this trait. DDR1 and DDHX1 are hub genes for miRNA regulatory networks controlling milk yield, while HHEX is an important transcription regulator for these networks. miR-18a, miR-221/222 cluster, and transcription factors HOXA7, and NOTCH 3 and 4, are important for the regulation of lactose. miR-142, miR-146a, and miR-EIA17-14144 (a novel miRNA), and transcription factors in the SMAD family and MYB, are important for the regulation of SCC. Important signaling pathways enriched for target genes of miRNAs of significant modules, included protein kinase A and PTEN signaling for milk yield, eNOS and Noth signaling for lactose, and TGF β, HIPPO, Wnt/β-catenin and cell cycle signaling for SCC. Relevant enriched gene ontology (GO)-terms related to milk and mammary gland traits included cell differentiation, G-protein coupled receptor activity, and intracellular signaling transduction. Overall, this study uncovered regulatory networks in which miRNAs interacted with each other to regulate lactation traits.

Nutritional strategies can decrease saturated fatty acids (SFAs) and increase health beneficial fatty acids (FAs) in bovine milk. The pathways/genes involved in these processes are not properly defined. Next-generation RNA-sequencing was used to investigate the bovine mammary gland transcriptome following supplemental feeding with 5 % linseed oil (LSO) or 5 % safflower oil (SFO). Holstein cows in mid-lactation were fed a control diet for 28 days (control period) followed by supplementation with 5 % LSO (12 cows) or 5 % SFO (12 cows) for 28 days (treatment period). Milk and mammary gland biopsies were sampled on days-14 (control period), +7 and +28 (treatment period). Milk was used to measure fat(FP)/protein(PP) percentages and individual FAs while RNA was subjected to sequencing. Milk FP was decreased by 30.38 % (LSO) or 32.42 % (SFO) while PP was unaffected (LSO) or increased (SFO). Several beneficial FAs were increased by LSO (C18:1n11t, CLA:10t12c, CLA:9c11t, C20:3n3, C20:5n3, C22:5n3) and SFO (C18:1n11t, CLA:10t12c , C20:1c11, C20:2, C20:3n3) while several SFAs (C4:0, C6:0, C8:0, C14:0, C16:0, C17:0, C24:0) were decreased by both treatments (P < 0.05). 1006 (460 up- and 546 down-regulated) and 199 (127 up- and 72 down-regulated) genes were significantly differentially regulated (DE) by LSO and SFO, respectively. Top regulated genes (≥2 fold change) by both treatments (FBP2, UCP2, TIEG2, ANGPTL4, ALDH1L2) are potential candidate genes for milk fat traits. Involvement of SCP2, PDK4, NQO1, F2RL1, DBI, CPT1A, CNTFR, CALB1, ACADVL, SPTLC3, PIK3CG, PIGZ, ADORA2B, TRIB3, HPGD, IGFBP2 and TXN in FA/lipid metabolism in dairy cows is being reported for the first time. Functional analysis indicated similar and different top enriched functions for DE genes. DE genes were predicted to significantly decrease synthesis of FA/lipid by both treatments and FA metabolism by LSO. Top canonical pathways associated with DE genes of both treatments might be involved in lipid/cholesterol metabolism. This study shows that rich α-linolenic acid LSO has a greater impact on mammary gland transcriptome by affecting more genes, pathways and processes as compared to SFO, rich in linoleic acid. Our study suggest that decrease in milk SFAs was due to down-regulation of genes in the FA/lipid synthesis and lipid metabolism pathways while increase in PUFAs was due to increased availability of ruminal biohydrogenation metabolites that were up taken and incorporated into milk or used as substrate for the synthesis of PUFAs.