Valine, a kind of branched-chain amino acid, plays a regulatory role beyond that of a building block in milk protein synthesis. However, the underlying molecular mechanism through which valine stimulates β-casein synthesis has not been clarified. Therefore, our study aimed to evaluate the effect of valine on β-casein synthesis and shed light into the molecular mechanism using an in vitro model. Results showed that valine supplementation significantly increased β-casein synthesis in bovine mammary epithelial cells (BMECs). Meanwhile, the supplementation of valine resulted in high levels of branched-chain aminotransferase transaminase 2 (BCAT2), TCA-cycle intermediate metabolites, and ATP, AMP-activated protein kinase (AMPK) inhibition, and mammalian target of rapamycin (mTOR) activation. Furthermore, the inhibition of BCAT2 decreased the β-casein synthesis and downregulated the AMPK-mTOR pathway, with similar results observed for AMPK activation. Together, the present data indicate that valine promotes the synthesis of β-casein by affecting the AMPK-mTOR signaling axis and that BCAT2-mediated valine catabolism is the key target.

The rumen is a vital organ containing vast amounts of microbes that play a key role in the digestion of nutrients and affect the production performance of ruminants. However, few studies have focused on the characterization of the ruminal microbiota composition and function in cows with long-term difference milk protein concentrations, and the relationship between milk protein concentration and ruminal microbiota remains elusive. In this study, we collected the data of milk protein concentrations of 1,025 Holstein cows for 10 months on a commercial farm. Based on the milk protein concentrations, 30 cows were selected and divided into three groups (n=10 per group): low milk protein group (LMP, milk protein concentration < 3.1 %), medium milk protein group (MMP, 3.1 % ≤ milk protein concentration < 3.4 %), and high milk protein group (HMP, milk protein concentration ≥ 3.4 %). The ruminal microbiome, metabolome, VFA concentrations and proportions, and amino acid profiles of the three groups were analyzed. The data showed that free amino acid (FAA) levels were lower in the rumen and higher in the plasma of HMP cows (P < 0.05). In addition, lower NH3 concentrations were observed in the rumen, plasma, and milk of the HMP cows (P < 0.05). Protease activity and isobutyric acid molar proportion in the rumen were lower in the HMP group (P < 0.05). Microbiome analysis showed that HMP cows had lower microbial diversity (represented as Shannon and Simpson indices) than LMP cows. At the genus level, lower relative abundances of Prevotella_1 and Ruminococcaceae_UCG_005 were observed in the HMP group (P < 0.05). At the operational taxonomic unit (OTU) level, a lower relative abundance of OTU3 (Prevotella ruminicola) was observed in the HMP group (P < 0.05). We found that the relative abundances of ruminal Prevotella_1 and OTU3 (Prevotella ruminicola) were negatively correlated with milk protein concentration (P < 0.05). These findings suggested that the cows with long-term high milk protein concentrations had lower microbial diversity and weaker protein degradation ability in the rumen. Furthermore, our observations identified a correlation between the milk protein concentration and ruminal microbiota.

Holstein cattle are well known for their high average milk yield but are more susceptible to disease and have lower fecundity than other breeds of cattle. The purpose of this study was to explore the relationship between ruminal metabolites and both milk performance and ruminal microbiota composition as a means of assessing the benefits of crossbreeding Montbéliarde and Holstein cattle. This experiment crossbred Holstein with Montbéliarde cattle, aimed to act as a reference for producing high-quality dairy products and improving the overall efficiency of dairy cattle breeding. Based on similar age, parity and lactation time, 46 cows were selected and divided into two groups (n  =  23 per group) for comparison experiment and fed the same formula: Montbéliarde×Holstein (MH, DIM  =  33.23  ±  5.61 d), Holstein (H, DIM  =  29.27  ±  4.23 d). Dairy herd improvement (DHI) data is an important basis for evaluating the genetic quality of bulls, understanding the quality level of milk, and improving feeding management. We collected the DHI data of these cows in the early lactation, middle lactation and late lactation period of 10  months. The results showed that the average milk production and protein content in Montbéliarde×Holstein were 1.76  kg (34.41  kg to 32.65  kg, p  >  0.05) and 0.1% (3.54 to 3.44%, p  <  0.05) higher than in Holstein cattle. Moreover, milk from Montbéliarde×Holstein cattle had lesser somatic cell score (1.66 to 2.02) than Holstein cattle (p  <  0.01). A total of 10 experimental cattle in early lactation were randomly selected in the two groups (Lactation time  =  92.70  ±  6.81), and ruminal fluid were collected by oral gastric tube. Using 16S rRNA microbial sequencing, we compared the ruminal microbiota composition and found that Montbéliarde×Holstein cattle had a lower abundance of Alphaproteobacteria (p  <  0.05) and higher abundance of Selenomonas than Holstein cattle (p  <  0.05). These bacteria play roles in protein degradation, nitrogen fixation and lactic acid degradation. The abundance of Succiniclasticum was also greater in Montbéliarde×Holstein cattle (p  =  0.053). Through ruminal metabolome analysis, we found that the levels of trans-ferulic acid, pyrrole-2-carboxylic acid, and quinaldic acid were significantly increased in Montbéliarde×Holstein cattle, while that of lathosterol was significantly decreased. The changes in the levels of these metabolites could confer improved antioxidant, anti-inflammatory, and antibacterial activities.