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The objective of this study was to determine the circulating microRNA (miRNA) profile in over-conditioned (HBCS) versus normal-conditioned (NBCS) dairy cows in combination with pathway enrichment analyses during the transition period. Thirty-eight multiparous Holstein cows were selected 15 wk before anticipated calving date based on their current and previous body condition scores (BCS) for forming either a HBCS group (n = 19) or a NBCS group (n = 19). They were fed different diets during late lactation to reach the targeted differences in BCS and backfat thickness until dry-off. A subset of 15 animals per group was selected based on their circulating concentrations of nonesterified fatty acids (on d 14 postpartum) and β-hydroxybutyrate (on d 21 postpartum), representing the greater or the lower extreme values within their BCS group. Blood serum obtained at d -49 and 21 relative to parturition (3 pools with 5 cows per each group and time point) were used to identify miRNA that were differentially expressed (DE) between groups or time points using miRNA sequencing. No DE-miRNA were discovered between NBCS versus HBCS. Comparing pooled samples from d -49 and d 21 resulted in 7 DE-miRNA in the NBCS group, of which 5 miRNA were downregulated and 2 miRNA were overexpressed on d 21 versus -49. The abundance of 5 of these DE-miRNA was validated in all individual samples via quantitative PCR and extended to additional time points (d -7, 3, 84). Group differences were observed for miR-148a, miR-122 as well as miR-455-5p, and most DE-miRNA (miR-148a, miR-122, miR-30a, miR-450b, miR-455-5p) were downregulated directly after calving. Subsequently, the DE-miRNA was used for bioinformatics analysis to identify putative target genes and the most enriched biological pathways. The most significantly enriched pathways of DE-miRNA were associated with cell cycle and insulin signaling as well as glucose and lipid metabolism. Overall, we found little differences in circulating miRNA in HBCS versus NBCS cows around calving.

The conjugated linoleic acid (CLA) isomers, a group of naturally occurring isomers of the essential fatty acid (FA) linoleic acid, have received special attention in animal and human nutrition. Although they have long been used as dietary integrators in dairy cows, the effects of CLA isomers on bovine immune cells remain mostly undisclosed. The present study aimed to cover this gap and investigate the in vitro effects of CLA on inflammatory functions, including chemotaxis, phagocytosis, killing capability, and extracellular respiratory burst of purified bovine monocytes (CD14+). The apoptosis rate of monocytes was addressed as well. Once assessed, the effects of different concentrations (10, 50, 100, and 500 μM) of the 2 main CLA isomers, namely cis-9,trans-11 and trans-10,cis-12, the experiments were carried out using a concentration of 50 μM of the CLA isomers, both individually and in a mixture (50:50). The immunomodulatory activities of linoleic acid, an essential FA, and stearic acid, a saturated FA, were also investigated. Only the 50:50 CLA mixture was able to reduce monocyte apoptosis and to increase the extracellular respiratory burst during experimental proinflammatory conditions, as assessed by measuring production of reactive oxygen species. Linoleic acid and CLA had no effects on chemotaxis, phagocytosis, or killing capability. Remarkably, treatment of monocytes with stearic acid significantly reduced their chemotactic capability. The present results demonstrated that CLA isomers do have immunomodulatory effects on some functions of bovine monocytes, and that the mixture of the 2 CLA isomers is more effective than the CLA isomers individually.

The identification of milk microbial communities in ruminants is relevant for understanding the association between milk microbiota and health status. The most common approach for studying the microbiota is amplifying and sequencing specific hypervariable regions of the 16S rRNA gene using massive sequencing techniques. However, the taxonomic resolution is limited to family and, in some cases, genus level. We aimed to improve taxonomic classification of the water buffalo milk microbiota by amplifying and sequencing the full-length 16S rRNA gene (1,500 bp) using Nanopore sequencing (single-molecule sequencing). When comparing with short-read results, we improved the taxonomic classification, reaching species level. We identified the main microbial agents of subclinical mastitis at the species level that were in accordance with the microbiological culture results. These results confirm the potential of single-molecule sequencing for in-depth analysis of microbial populations in dairy animals.