Background: MicroRNAs (miRNAs) have been implicated in the regulation of milk protein synthesis and development of the mammary gland (MG). However, the specific functions of miRNAs in these regulations are not clear. Therefore, the elucidation of miRNA expression profiles in the MG is an important step towards understanding the mechanisms of lactogenesis. Results: Two miRNA libraries were constructed from MG tissues taken from a lactating and a non-lactating Holstein dairy cow, respectively, and the short RNA sequences (18–30 nt) in these libraries were sequenced by Solexa sequencing method. The libraries included 885 pre-miRNAs encoding for 921 miRNAs, of which 884 miRNAs were unique sequences and 544 (61.5%) were expressed in both periods. A custom-designed microarray assay was then performed to compare miRNA expression patterns in the MG of lactating and non-lactating dairy cows. A total of 56 miRNAs in the lactating MG showed significant differences in expression compared to non-lactating MG (P<0.05). Integrative miRNA target prediction and network analysis approaches were employed to construct an interaction network of lactation-related miRNAs and their putative targets. Using a cell-based model, six miRNAs (miR-125b, miR-141, miR-181a, miR-199b, miR-484 and miR-500) were studied to reveal their possible biological significance. Conclusion: Our study provides a broad view of the bovine MG miRNA expression profile characteristics. Eight hundred and eighty-four miRNAs were identified in bovine MG. Differences in types and expression levels of miRNAs were observed between lactating and non-lactating bovine MG. Systematic predictions aided in the identification of lactation-related miRNAs, providing insight into the types of miRNAs and their possible mechanisms in regulating lactation.

The mammary epithelial cells (MECs) of high‐producing dairy cows are likely to be subject to oxidative stress (OS) due to the intensive cell metabolism. The objectives of this study were to investigate the cytoprotective effects of resveratrol against hydrogen peroxide‐ (H₂O₂‐) induced OS in cultured bovine MECs (MAC‐T). Pretreatment of MAC‐T cells with resveratrol could rescue the decrease in cell viability and resulted in lower intracellular reactive oxygen species (ROS) accumulation after H₂O₂ exposure. Resveratrol helped MAC‐T cells to prevent H₂O₂‐induced endoplasmic reticulum stress and mitochondria‐related cell apoptosis. Moreover, resveratrol induced mRNA expression of multiple antioxidant defense genes in MAC‐T cells under normal/oxidative conditions. Nuclear factor erythroid 2‐related factor 2 (Nrf2) was required for the cytoprotective effects on MAC‐T cells by resveratrol, as knockdown of Nrf2 significantly abolished resveratrol‐induced cytoprotective effects against OS. In addition, by using selective inhibitors, we further confirmed that the induction of Nrf2 by resveratrol was mediated through the prolonged activation of PI3K/Akt and ERK/MAPK pathways but negatively regulated by p38/MAPK pathway. Overall, resveratrol has beneficial effects on bovine MECs redox balance and may be potentially used as a therapeutic medicine against oxidative insult in lactating animals.

Lactation is extremely important for dairy cows; however, the understanding of the underlying metabolic mechanisms is very limited. This study was conducted to investigate the inherent metabolic patterns during lactation using the overall biofluid metabolomics and the metabolic differences from non-lactation periods, as determined using partial tissue-metabolomics. We analyzed the metabolomic profiles of four biofluids (rumen fluid, serum, milk and urine) and their relationships in six mid-lactation Holstein cows and compared their mammary gland (MG) metabolomic profiles with those of six non-lactating cows by using gas chromatography-time of flight/mass spectrometry. In total, 33 metabolites were shared among the four biofluids, and 274 metabolites were identified in the MG tissues. The sub-clusters of the hierarchical clustering analysis revealed that the rumen fluid and serum metabolomics profiles were grouped together and highly correlated but were separate from those for milk. Urine had the most different profile compared to the other three biofluids. Creatine was identified as the most different metabolite among the four biofluids (VIP = 1.537). Five metabolic pathways, including gluconeogenesis, pyruvate metabolism, the tricarboxylic acid cycle (TCA cycle), glycerolipid metabolism, and aspartate metabolism, showed the most functional enrichment among the four biofluids (false discovery rate < 0.05, fold enrichment >2). Clear discriminations were observed in the MG metabolomics profiles between the lactating and non-lactating cows, with 54 metabolites having a significantly higher abundance (P < 0.05, VIP > 1) in the lactation group. Lactobionic acid, citric acid, orotic acid and oxamide were extracted by the S-plot as potential biomarkers of the metabolic difference between lactation and non-lactation. The TCA cycle, glyoxylate and dicarboxylate metabolism, glutamate metabolism and glycine metabolism were determined to be pathways that were significantly impacted (P < 0.01, impact value >0.1) in the lactation group. Among them, the TCA cycle was the most up-regulated pathway (P < 0.0001), with 7 of the 10 related metabolites increased in the MG tissues of the lactating cows. The overall biofluid and MG tissue metabolic mechanisms in the lactating cows were interpreted in this study. Our findings are the first to provide an integrated insight and a better understanding of the metabolic mechanism of lactation, which is beneficial for developing regulated strategies to improve the metabolic status of lactating dairy cows.

L-lysine (Lys) is a critical dietary nutrient for mammary gland development and milk production. However, the specific pathways of Lys utilization and how milk protein synthesis is affected in bovine mammary epithelial cells (BMECs) are poorly understood. We aimed to investigate the effects of Lys on milk protein synthesis and the mechanism of Lys uptake and catabolism in BMECs. BMECs were cultured in 0, 0.5, 1.0, 1.5, 2.0, 5.0, and 10.0 mmol Lys/L to detect cell viability, or cultured in 0-2.0 mmol Lys/L with l-[ring-3H5] phenylalanine to study the effect of Lys on protein turnover, or cultured in Krebs buffer with [U-14C] l-Lys to quantify Lys metabolism. In some experiments, BMECs were cultured in a conditioned medium alone or including 1.0 mmol Lys/L and 2-amino-endo-bicyclo [2.2.1] heptane-2-carboxylic acid (BCH) for 24 h to analyze the expression of amino acid transporter B (0+) (ATB0,+), mammalian target of rapamycin (mTOR), and Janus kinase 2 (JAK2)-signal transducer and activator of transcription 5 (STAT5) pathways. Including 1.0 mmol Lys/L in cultures increased cell viability by 17-47% and protein synthesis by 7-23%, whereas protein degradation was inhibited by 4-64% compared with BMECs cultured with 0, 0.5, or 2.0 mmol Lys/L (all P ≤ 0.05). Studies that used [U-14C] l-Lys showed that most Lys was incorporated into proteins (90%), whereas the remainder was either oxidized into CO2 (4%) or used as a substrate for aspartate (3%) and histidine synthesis (3%). Furthermore, Lys significantly increased expression of ATB0,+ (71% mRNA and 44% protein), STAT5 (27% mRNA and 21% phosphorylated proteins), and mTOR (51% mRNA and 22% phosphorylated proteins) compared with cells without Lys. Lys promoted protein synthesis, mostly through enhancing uptake by ATB0,+ and the mTOR and JAK2-STAT5 pathways. Understanding the utilization of Lys in BMECs provides insights into the role of amino acid nutrition in bovine milk production.