Heat stress (HS) is one of the most serious factors to negatively affect the lactation performance of dairy cows. Bovine mammary epithelial cells are important for lactation. It was demonstrated that HS decreases the lactation performance of dairy cows, partly through altering gene expression within bovine mammary epithelial tissue. However, the cellular metabolism mechanisms under HS remains largely unknown. The objective of this study was to determine whether HS induced changes in intracellular metabolites and gene transcription related to amino acid metabolism, amino acid transportation and the mTOR signaling pathway. Immortalized bovine mammary epithelial cell lines (MAC-T cells, n = 5 replicates/treatment) were incubated for 12 h at 37 °C (Control group) and 42 °C (HS group). Relative to the control group, HS led to a greater mRNA expression of heat shock protein genes HSF1, HSPB8, HSPA5, HSP90AB1 and HSPA1A. Compared with the control group, metabolomics using liquid chromatography tandem–mass spectrometry identified 417 differential metabolites with p < 0.05 and a variable importance in projection (VIP) score >1.0 in the HS group. HS resulted in significant changes to the intracellular amino acid metabolism of glutathione, phenylalanine, tyrosine, tryptophan, valine, leucine, isoleucine, arginine, proline, cysteine, methionine, alanine, aspartate and glutamate. HS led to a greater mRNA expression of the amino acid transporter genes SLC43A1, SLC38A9, SLC36A1, and SLC3A2 but a lower mRNA expression of SLC7A5 and SLC38A2. Additionally, HS influenced the expression of genes associated with the mTOR signaling pathway and significantly upregulated the mRNA expression of mTOR, AKT, RHEB, eIF4E and eEF2K but decreased the mRNA expression of TSC1, TSC2 and eEF2 relative to the control group. Compared with the control group, HS also led to greater mRNA expression of the CSN1S2 gene. Overall, our study indicates that bovine mammary epithelial cells may have the ability to resist HS damage and continue milk protein synthesis partly through enhanced intracellular amino acid absorption and metabolism and by activating the mTOR signaling pathway during HS.

Atherosclerosis (AS) is recognized as the original cause of most cardiovascular and cerebrovascular diseases. The dual-protein (DP) nutrition that consists of soy protein and whey protein is reported to be associated with a reduction in AS; however, the relationship between DP and AS remains ambiguous. Therefore, this study aimed to verify the effect of DP on AS and explore the optimal DP intake to improve AS. ApoE^−/− mice were administrated with low- (LDP), middle- (MDP), and high-dose (HDP) DP. The MDP group exhibited significant improvements in AS. In terms of lipid metabolism, the levels of plasma total triglyceride and LDL-C and the mRNA expression levels of Cyp7a1 and PCSK9 were markedly tuned in the MDP group. In addition, the MDP treatment group had a substantially lower inflammatory response and better intestinal barrier function than LDP and HDP groups. The species richness demonstrated by the Chao1 index was distinctly increased in the MDP group, and the relative abundance of intestinal-permeability-protective microbes Blautia and Akkermansia was significantly elevated. In summary, an adequate intake of DP was able to counteract atherosclerosis development in ApoE^−/− mice, and this study provides a scientific theoretical basis for the application of DP in the food and pharmaceutical fields.

Winter wheat (Triticum aestivum L.) silage has high feeding value and has become an important roughage resource in China. To recognize the optimal fermentation time of the silage product, this study systematically evaluated the temporal dynamics of microbial communities and metabolic profiles in whole winter wheat silage at days 7, 14, 30, 50, and 70. The dry matter (DM) content slightly fluctuated with the extension of fermentation time, with 28.14% at 70 days of ensiling. The organic matter and neutral detergent fiber content gradually decreased with the extension of fermentation time. A significant decrease in pH was observed at days 30, 50, and 70 compared to days 7 and 14 (p < 0.05), with the lowest pH value of 4.4 recorded at day 70. The contents of lactic acid, acetic acid, butyric acid, and total volatile fatty acids gradually increased with the extension of fermentation time, reaching a maximum at 70 days of ensiling. The dominant bacteria were Proteobacteria and Firmicutes at the phylum level, and the predominant bacteria were Hafnia-Obesumbacterium, Enterobacter, and Lactobacillus at the genus level. The relative abundance of Hafnia-Obesumbacterium and Lactobacillus fluctuated slightly with the duration of fermentation, reaching a minimum for the former and a maximum for Lactobacillus at 50 days of ensiling. By day 70, Sporolactobacillus emerged as a distinct silage biomarker. The dominant fungi was Ascomycota at the phylum level, and the predominant fungi were Fusarium and an unidentified fungus at the genus level. The correlation analysis revealed significant pH–organic acid–microbe interactions, with pH negatively correlating with organic acids but positively with specific bacteria, while organic acids showed complex microbial associations. Collectively, under natural fermentation conditions, the optimal fermentation period for wheat silage exceeds 70 days, and Sporolactobacillus shows potential as a microbial inoculant for whole winter wheat silage. These findings provide a theoretical foundation for optimizing whole winter wheat silage utilization and enhancing fermentation quality.