Dietary fiber enhances milk yield in plateau dairy cows via activation of the rumen microbiota-mammary gland axis

Abstract

Milk yield in high-altitude regions such as the Qinghai-Tibet Plateau is low due to hypoxic stress and impaired mammary gland function. This study aims to determine whether a fiber-supplemented diet could increase milk yield in plateau dairy cows through modulating rumen microbiota and downstream metabolic signaling. Holstein cows were assigned to diets containing either Brassica rapa L. or an aquatic plant with a high neutral and acid-detergent fiber content. Milk yield and rumen metabolites were analyzed, and additional functional assays were performed using bovine mammary epithelial cells (BMECs) cultured under hypoxic conditions. The Brassica rapa L. supplementation significantly increased milk yield, which was associated with elevated levels of fiber-derived metabolites, including cholesterol valerate and 5-oxoeicosapentaenoic acid. These metabolites activated liver X receptor signaling in mammary cells under hypoxia, as validated by proteomic analysis and LXRα expression. Gene enrichment analysis revealed that LXR signaling was associated with lipid metabolism and cellular adaptation to low oxygen. These results support a fiber-microbiota-mammary axis, showing that fiber supplementation enhances milk yield through metabolic signaling. Moreover, this study presents a sustainable and feasible method to enhance milk production in ruminants under environmental stress.

Keywords: LXR signaling; fiber; hypoxia; mammary gland; milk yield.

Figure 1

Dietary fiber from Brassica rapa L. improves milk yield in plateau dairy cows. (A) Nutritional composition of the control, Brassica rapa L., and aquatic plant dietss. (B) Daily milk yield in cows fed each diet. Brassica rapa L. and aquatic plant diets significantly increased milk production compared to the control group (p < 0.05), with the Brassica rapa L. group showing the highest yield. Data are presented as mean ± SEM. * p < 0.05; ** p < 0.01.

Figure 2

Rumen metabolomic profiling reveals increased lipid-associated signaling metabolites under fiber feeding. (A) Volcano plot showing differentially abundant lipid metabolites between Brassica rapa and control groups. Red and blue dots represent significantly upregulated and downregulated metabolites, respectively. (B) Metabolic network highlighting the association between lipid metabolites and liver X receptor alpha (LXRα), with connections to oxidized lipids, cholesterol, and downstream effectors such as SREBP-1c. (C) Boxplots showing significantly elevated concentrations of 5-oxoeicosapentaenoic acid (5-OxoEPA), cholesterol valerate, and docosahexaenoic acid-d5 (DHA-d5) in the Brassica rapa group (* p < 0.05).

Figure 3

Hypoxia reprograms mammary epithelial cell proteome with enrichment of LXR-related lipid pathways. (A) Volcano plot showing significantly up- and downregulated proteins. (B) KEGG pathway enrichment of differentially expressed proteins under normoxia and hypoxia. (C) GO enrichment analysis highlights changes in lipid metabolism, circadian regulation, and VHL complex components. Significance threshold: fold change >1.5, p < 0.05.

Figure 4

Dietary metabolites and hypoxia synergistically induce LXRα expression and localization. (A) Protein interaction network showing LXRα as a central node connecting fatty acid metabolism, circadian rhythm, and hypoxia response pathways. (B) Western blot analysis of LXRα expression in BME-UV1 cells under normoxia, hypoxia, at 4 h, 8 h, and 12 h following CoCl₂ exposure. (C) Immunofluorescence images of GFP-tagged LXRα showing nuclear localization under hypoxic conditions. (D) Correlation analysis of the TCGA-BRCA dataset showing a significant positive correlation between hypoxia activity score and NR1H3 (LXRα) expression. (E) Co-immunoprecipitation (Co-IP) reveals a direct interaction between LXRα and HIF-1α under hypoxic conditions.

Figure 5

A mechanistic model associates fiber intake with lactation via a microbiota-mammary axis. (A) Volcano plot of differentially expressed genes in HEK293 cellss overexpressing bovine LXRα. (B) KEGG pathway enrichment of these genes shows involvement in cholesterol metabolism, fatty acid biosynthesis, PPAR signaling, and bile secretion. (C) Schematic model of the proposed “rumen microbiota–mammary gland axis.” This is a conceptual summary based on earlier metabolomic and proteomic findings in this study, rather than a presentation of new microbiota sequencing data.