Mastitis in dairy cows is an important factor restricting the healthy development of dairy industry. Natural extracts have become a research hotspot to alleviate and prevent diseases because of their unique properties. The purpose of this study was to investigate the effects of resveratrol (RES) on the mitochondrial biosynthesis, antioxidation, and anti-inflammatory in bovine mammary epithelial cells (BMECs) and its mechanism involved. Blood samples were collected from six healthy cows and six mastitis affected cows, respectively, and lipopolysaccharide (LPS) was used to treat BMECs to construct inflammation models, gene interference is achieved by transfection. The results showed that messenger RNA (mRNA) expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) was down-regulated and mitochondrial biogenesis-related gene expression was disrupted in the blood of mastitis cows and LPS-induced BMECs. RES is the best active substance to activate PGC-1α. The addition of RES can effectively alleviate the production of BMECs reactive oxygen species (ROS) and mitochondrial damage induced by LPS, and improve the antioxidation and anti-inflammatory ability, while the alleviation effect of RES is inhibited after interfering with protein kinase AMP-activated catalytic subunit α 1 (PRKAA1). In summary, our study emphasizes that PRKAA1 is a key gene mediating the activation of PGC-1α by RES, which regulates mitochondrial biosynthesis, inhibits ROS release, attenuates mitochondrial damage, and improves mitochondrial antioxidant capacity through the activation of PGC-1α by PRKAA1, thus attenuating the inflammatory response in BMECs.

One hundred and thirteen mid-lactation cows fed same diets and supplemented with 20 g/d rumen-protected methionine (RPM) for 8 weeks were used to investigate the individual responses of dairy cows to RPM in terms of lactation performance, amino acids (AA) metabolism, and milk metabolites. Among the cows, 10 cows exhibited positive responses (PR) and 10 cows showed limited responses (LR) in energy-corrected milk yield to RPM were used for further analysis. The lactation performance changed from gradual decline to steady increase in PR cows, while kept downward trend in LR cows following RPM supplementation. In PR cows, the AA metabolism was notably enhanced after RPM supplementation, evidenced by increased mammary blood flow (69.4%, P = 0.05), mammary uptake and clearance rate and uptake-to-output ratio (U:O) of essential AA. The improved AA metabolism could be attributed to the enrichment of pyrimidine (P = 0.06) and pyruvate (P = 0.07) metabolism pathways, which may have stimulated mammary cell proliferation and enhanced AA uptake. Additionally, the upregulation of milk biotin (fold change > 2, variable importance projection > 1), known to support milk yield, likely contributed to the PR observed in PR cows. Conversely, in LR cows, RPM supplementation did not improve AA metabolism, decrease was observed in mammary uptake, mammary clearance rate, and U:O of cysteine, potentially due to cysteine being irreversibly converted from methionine. Moreover, the enrichment of central carbon metabolism in cancer pathway (P = 0.06), which also utilize methionine, along with the lysine degradation pathway (P = 0.04), suggests that methionine in the mammary glands may have been diverted toward non-lactational metabolic processes, resulting in absence of PR in LR cows. Our results indicate that the responses to RPM in dairy cows are individualized, with variation in lactation performance likely driven by differential AA metabolism.

We previously reported changes in gene expression in mammary tissue from non-inflamed mammary glands adjacent to an inflamed gland challenged with lipopolysaccharide (LPS). We determined if changes in the expression of selected genes in non-inflamed glands would be replicated in RNA isolated from milk fat. Cows were milked twice daily prior to experiment. Per cow, one mammary gland (QLPS) was randomly assigned to receive an intramammary infusion of 50 µg LPS immediately after morning milking on d-0. The ipsilateral (QI) and contralateral (QC) mammary glands adjacent to QLPS remained untreated. Quarter foremilk samples from all mammary glands were collected on d-1 and d-0 for milk composition and isolation of RNA for quantification of selected genes via quantitative polymerase chain reaction. Symptoms of clinical mastitis developed only in QLPS and were apparent within 3 h post-challenge. In QI and QC, lactose percentages were lower at 12 h post-challenge compared to d-1, but milk fat and protein contents were not different. For gene expression, 7 of 13 selected genes were differentially regulated in non-inflamed glands. In QI but not QC, LALBA expression was lower at 12 h post-challenge than on d-1. One gene of interest, LPIN1, was significantly upregulated in QI and QC but downregulated in QLPS at 12 h post-challenge. Five additional immune or stress-related genes were significantly upregulated in QLPS and, to a lesser but significant degree, in QI and QC compared to d-1. Notably, expression of two immune genes (NFKBIA, PTX3) was significantly greater in QI than QC despite QI having a numerically lower somatic cell count. Minor changes in the composition of milk secreted by non-inflamed mammary glands were linked to several immune and stress responses in those glands. Further, individual non-inflamed mammary glands responded differently depending on their position relative to the mastitic gland.

Milk fat is a high-value component of the U.S. dairy market. It is the major energy component of milk and is responsible for many organoleptic and technological characteristics of milk and dairy products. In addition, milk fat is unquestionably distinctive among all dietary fats that humans consume, as it is not only comprised of several hundred different fatty acids (FAs) but also contains a wide and unique array of bioactive lipids. Milk fat is dispersed in milk primarily in the form of fat globules. These cytoplasmic lipid droplets originate from mammary epithelial cells (MECs) and are secreted into the alveolar lumen surrounded by a membrane. Many advances in our knowledge of specific enzymes involved in milk lipid synthesis, the selectivity of the triacylglyceride (TAG) synthesis enzymes for specific FAs, the molecular mechanisms behind the uptake of long-chain FAs into the cells and the milk lipid secretion process have led to an improved understanding of the biology of milk fat synthesis. However, research to provide deeper insights into the mechanism of lipid synthesis in MECs is warranted and might lead to novel strategies to alter milk fat content and quality to benefit the dairy industry and meet dietary recommendations and consumer demands for foods that positively impact health. In this review, we aimed to provide a general overview of our current knowledge of the molecular aspects of milk lipid synthesis in MECs, from the uptake of blood-derived precursors to the intracellular formation of TAG-rich fat droplets secreted into milk as milk fat globules. We also highlight some current gaps in the knowledge that warrant further exploration. Given the importance of dairy food in the human diet, a better understanding of these processes could help develop novel strategies to alter milk fat composition in ways that benefit both human health and dairy producers.

Rumen microbial biohydrogenation (RBH) is the major factor responsible for the bovine milk rich in saturated fatty acids (FAs). Here, we evaluated the effects of nutritional manipulation of ruminal propionogenesis and methanogenesis, two primary hydrogen sinks, on the RBH and milk FA profiles in vivo and in vitro using three propionogenesis enhancers (fumarate [FUM], biotin and monensin) and one methanogenesis inhibitor (N-[2-(nitrooxy)ethyl]-3-pyridinecarboxamide [NPD]). The in vivo results showed that inclusion of FUM in lactating dairy goat diet could protect dietary unsaturated FAs against RBH with increased proportions of C18:2n − 6 (by 33.5%), C18:3n − 3 (by 38.1%) and RBH intermediates (e.g. trans-10 C18:1 and trans-11 C18:1) in rumen contents. Additionally, FUM supplementation increased the milk Δ9 desaturase index (by 15.5%) with higher cis-9 monounsaturated FAs in the milk. As a result, FUM increased the proportions of polyunsaturated and monounsaturated FAs in the milk with lower atherogenicity index (by −15.3%) and thrombogenicity index (by −19.5%). Conversely, supplementing NPD increased RBH completeness (by 7.4%) with higher milk atherogenicity index (by 10.5%) and thrombogenicity index (by 8.7%). The adverse effects of NPD on the milk FA profiles can be eliminated when supplemented in combination with FUM. The metagenomic analyses showed that neither FUM nor NPD affect the rumen microbial α- or β-diversity at the strain or gene level. The in vitro study showed that the conversion rate of FUM to propionate was increased from 54.7% to 80.6% when FUM supplemented in combination with biotin and monensin, resulting a higher anti-RBH potential. Accordingly, manipulation of ruminal methanogenesis and propionogenesis can redirect hydrogen toward or away from RBH and thereby influence the milk FA profiles. FUM is a promising feed additive in ruminant not only to reduce the methane emissions as previously proved but also to improve the nutritional desirability of the milk FA profiles for human health.

Somatic cells (SCs) in milk are a heterogeneous population composed of several subsets of cells. However, a complete understanding of this heterogeneity in cow’s milk remains elusive. This study aimed to characterize heterogeneity within mammary epithelial (MEC) and immune cell subpopulations from healthy Holstein cows. An initial cell characterization of SC populations was completed using a single milk collection (3.8 L) from a base population of 25 multiparous Holstein cows to identify MEC and immune cells using flow cytometry with Butyrophilin 1A1 (BTN) and CD45 as cell surface markers. From the base population, 5 multiparous cows (≥300 days in milk (DIM), ≤162 × 103 SC/mL, and milk yield (MY) ≥ 25 kg/d) were selected for fluorescence activated cell sorting and single-cell RNA sequencing (scRNA-seq) analysis. A single-cell-suspension of approximately 1,000 sorted cells was prepared from each cow for characterization using scRNA-seq. Gel beads and barcodes were generated, cDNA amplified, cDNA sequencing libraries constructed and sequenced. After data normalization, scaling, and filtering control, two CD45+ databases were generated. The CD45+ databases contained 923 and 851 single cells, each comprising 17,771 and 12,156 features, respectively. Principal component analysis revealed seven and eight distinguishing clusters. Based on marker expression, most immune cells present in the samples were T cells (CD3E and PTPRC). Three different T cell subpopulations were revealed: helpers (CD4), cytotoxic (CD8A and CD8B), and regulatory T cells (IL2RA). The remaining four clusters were composed of granulocytes (neutrophils, eosinophils, and basophils; TLR4 and CXCL8), macrophages (PTPRC, CD14, CD68, TL2, IL1B), and a small population of B cells (CD19, CD22, and MS4A1). The study and characterization of immune cell subpopulations present in milk provide a basis for developing greater insights into mammary gland immune function, offering potential avenues for enhancing animal health and milk production in the future.