Arginine (Arg), as an important functional amino acids (AA), is essential for milk protein synthesis in lactating ruminants. Arg shares transporters with cationic and neutral AA in mammary epithelial cells. Therefore, competitive inhibition might exist among these AA in uptake by mammary epithelial cells. In this study, cultured bovine mammary epithelial cells (BMEC) was used as the model to investigate whether the availability of L-Arg (0.7, 1.4, 2.8, 5.6 and 11.2 mM) affects the uptake of other AA and if this related to α_S1-casein synthesis, and whether Arginine-Arginine (Arg-Arg) substituting part of free L-Arg can alleviate competitive inhibition among Arg and other AA, so as to promote α_S1-casein synthesis. Our results showed 2.8 mM L-Arg generated the greatest positive effects on α_S1-casein synthesis and the activation of mammalian target of rapamycin (mTOR) signaling pathway (P < 0.01). With L-Arg supply increasing from 0.7 to 11.2 mM, the net-uptake of other AA (except Glu and Ala) decreased linearly and quadratically (P_linear < 0.01; P_quadratic < 0.01). Compared with 2.8 mM, the net-uptake of essential amino acids (EAA) and total amino acids (TAA) were lower at 11.2 mM L-Arg group, while greater at 1.4 mM L-Arg group (P < 0.01). Arg-Arg dipeptide replacing 10% free L-Arg increased α_S1-casein synthesis (P < 0.05), net-uptake of EAA and TAA, as well as phosphorylation level of mTOR and p70 ribosomal protein S6 kinase (P70S6K) and mRNA expression of oligopeptide transporter 2 (PepT2; P < 0.01). These observations suggested that the increased α_S1-casein synthesis by 10% Arg-Arg dipeptide might be related to the increase of AA availability and the activation of mTOR signaling pathway in BMEC.

The purpose of this research was to investigate the impact of dietary supplementation of Caragana korshinskii tannin (CKT) on rumen fermentation, methane emission, methanogen community and metabolome in rumen of sheep. A total of 15 crossbred sheep of the Dumont breed with similar body conditions, were divided into three groups (n = 5), which were fed with CKT addition at 0, 2 and 4%/kg DM. The study spanned a total of 74 days, with a 14-day period dedicated to adaptation and a subsequent 60-day period for conducting treatments. The results indicated that the levels of ammonia nitrogen (NH₃-N) and acetate were reduced (p < 0.05) in rumen sheep fed with 2 and 4% CKT; The crude protein (CP) digestibility of sheep in 2 and 4% CKT groups was decreased(p < 0.05); while the neutral detergent fiber (NDF) digestibility was increased (p < 0.05) in 4% CKT group. Furthermore, the supplementation of CKT resulted in a decrease (p < 0.05) in daily CH₄ emissions from sheep by reducing the richness and diversity of ruminal methanogens community, meanwhile decreasing (p < 0.05) concentrations of tyramine that contribute to methane synthesis and increasing (p < 0.05) concentrations of N-methy-L-glutamic acid that do not contribute to CH₄ synthesis. However, CH₄ production of DMI, OMI, NDFI and metabolic weight did not differ significantly across the various treatments. To sum up, the addition of 4% CKT appeared to be a viable approach for reducing CH₄ emissions from sheep without no negative effects. These findings suggest that CKT hold promise in mitigating methane emissions of ruminant. Further investigation is required to evaluate it effectiveness in practical feeding strategies for livestock.

Under current dairy cow feeding conditions, the conversion efficiency of dietary nitrogen into milk protein is less than 30%. A thorough understanding of amino acids (AA) uptake and utilization in mammary in response to AA supply is essential for accurately modeling nutritional requirements. This study utilized bovine mammary epithelial cells (BMEC) as a model to investigate the effects of increasing total essential amino acids (EAA) concentrations (0.00, 0.97, 1.94, 3.88, and 7.76 mM) on AA uptake, metabolism, and casein synthesis. The control group (0.00 mM) contained the basal medium without any additional EAA supplementation. The EAA mixture consisted of Thr, Lys, Met, His, Arg, Leu, Ile, Val, and Phe, maintained at fixed relative proportions that matched those found in the mammary artery of dairy cows at peak lactation. Nine cell culture plates were used per treatment, with 6 plates for casein expression analysis and 3 plates for proteomics analysis. After 24 h of incubation with the EAA, cells were harvested for the respective analyses. The culture medium from 6 plates was collected for individual AA content measurement. Data were analyzed using SAS (version 9.4). Orthogonal polynomial contrasts were used to determine the linear, quadratic, and cubic effects of treatments. Significance was declared at P < 0.05. As EAA concentrations in the BMEC medium increased, protein expression of αS1-casein, β-casein, and κ-casein, as well as total EAA, total AA, and each individual EAA uptake (except for Lys) increased quadratically (Pquadratic < 0.01). Meanwhile, the uptake efficiency of total EAA, Thr, Met, Ile, Phe, and His decreased linearly (Plinear < 0.01). Moreover, with increasing total EAA concentration from 0.97 to 7.76 mM, decreases in the uptake efficiency of Thr, Ile, and Phe were greater than for other EAA. Lysine uptake efficiency showed no difference among EAA-treated groups (P > 0.05). The protein expression of key enzymes involved in Met catabolism exhibited significant decreases with increasing EAA concentration (Plinear < 0.01). In contrast, The protein expression of key enzymes associated with Lys (Plinear = 0.008), branched-chain amino acids (Pquadratic < 0.05), and Arg (Plinear < 0.001 or Pquadratic < 0.001) catabolism demonstrated opposite trends, showing significant increases with increasing EAA concentration. These findings suggest that when EAA supply exceeds BMEC metabolic requirements, further increases in EAA supply reduce EAA utilization efficiency without enhancing casein synthesis. Furthermore, individual EAA demonstrate distinct uptake patterns and catabolic responses to changes in total EAA concentration.

Glucose has been demonstrated to affect milk protein synthesis in dairy cows. However, its potential mechanisms has not been thoroughly studied. The objective of this study was to investigate the effects of glucose availability on α_S1-casein synthesis, glucose uptake, metabolism, and the expression of proteins involved in AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway in bovine mammary epithelial cells (BMEC). BMEC were treated for 24 h with different concentrations of glucose (0, 7, 10.5, 14, 17.5, and 21 mM). The results showed that 10.5 and 14 mM glucose supply increased the expression of α_S1-casein, glucose uptake, cellular ATP content, and the phosphorylation of mTOR and P70S6K, but repressed AMPK phosphorylation in BMEC. Compared with 10.5 and 14 mM glucose supply, 17.5 and 21 mM glucose decreased the expression of α_S1-casein, P70S6K phosphorylation as well as the activity of hexokinase (HK) and pyruvate kinase (PK), but increased the activity of glucose-6-phosphate dehydrogenase (G6PD). These results indicate that 10.5 to 14 mM glucose supply is the proper range for α_S1-casein synthesis, and the promotion effects may be related to the increase of glucose uptake, ATP content and the changes of key proteins’ phosphorylation in AMPK/mTOR signaling pathway. However, the inhibition of the expression of α_S1-casein by 17.5 and 21 mM glucose may be associated with the changes of key enzymes’ activity involved in glucose metabolism. Glucose play an important role in milk protein synthesis in dairy cows. But the effects of glucose availability on casein synthesis and its underlying mechanisms has not been thoroughly studied. To elucidate the underlying mechanisms of glucose availability affecting casein synthesis, the effects of glucose availability on α_S1-casein synthesis, glucose uptake, metabolism, and the expression of proteins involved in AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway in bovine mammary epithelial cells were measured. We found that the expression of α_S1-casein increased with 10.5 and 14 mM glucose supplementation, which may be associated with the increase of glucose uptake, ATP content and the changes of key proteins’ phosphorylation in AMPK/mTOR signaling pathway. The inhibition of α_S1-casein expression with 17.5 and 21 mM glucose supplementation may be related to the changes of key enzymes’ activity involved in glucose metabolism. This study provided an insight into the potential mechanisms of glucose availability affecting milk protein synthesis.

Chestnut tannin (CHT) exhibits multiple biological activities, including antioxidant, antimicrobial, and anti-inflammatory properties. It can modulate the rumen microbiota composition, promoting animal health and improving production performance. This study investigates the effects of CHT on ruminal fermentation, cellulolytic activity, and microbial community of sheep in vivo and in vitro experiments. Eighteen 1.5-yr-old Mongolian rams fitted with permanent rumen cannulas (initial body weight: 43.0 ± 2.0 kg) were ranked by body weight from lowest to highest and then divided into six blocks, with three sheep per block. Within each block, the sheep were randomly assigned to one of the following dietary treatments: 1) control—basal diet without CHT (0%), 2) 2% CHT—basal diet supplemented with CHT at 2% DM, and 3) 6% CHT—basal diet supplemented with CHT at 6% DM. Rumen fluid was collected to evaluate fermentation parameters, while rumen contents were analyzed for cellulase activity, total bacterial community, and cellulolytic bacterial populations. Neutral detergent fiber (NDF) degradability was measured using the nylon bag technique. In vitro, dominant ruminal cellulolytic bacteria were cultured with 200 μg/mL and 600 μg/mL CHT to evaluate their growth, morphology, enzyme activities, and cellulose degradation capacity. In vivo results indicated that 6% CHT significantly reduced growth performance and the abundance of cellulolytic bacteria (P < 0.05). The concentration of NH3-N, in situ degradability of NDF, and xylanase activity decreased with increasing CHT supplementation (P < 0.05). In vitro results demonstrated that 200 μg/mL and 600 μg/mL CHT inhibited the growth of fiber-degrading bacteria, disrupted bacterial morphology, reduced the degradation rate of cellulolytic bacteria, and suppressed fiber-degrading enzyme activity (P < 0.05). Collectively, CHT reduced the population of cellulolytic bacteria in the rumen of sheep, inhibited cellulase activity, and affected cellulose digestion in sheep. These findings demonstrate that CHT supplementation inhibits ruminal fiber degradation through direct suppression of cellulolytic bacterial growth and activity.

The efficiency of phenylalanine (Phe) utilization for milk protein synthesis in dairy cows is limited, and its uptake and metabolic mechanisms in the mammary tissue remaining unclear. This study investigated the effects of Phe availability (0.07, 0.14, 0.28, and 0.56 mM) on amino acid metabolism and casein synthesis in bovine mammary epithelial cells (BMECs) cultured for 24 h. Results showed that α_S1-casein, β-casein, and κ-casein expression peaked at 0.14 mM Phe (p < 0.05). At this optimal concentration, amino acid transporters (SLC7A5, SLC7A8, and SLC38A2) were upregulated, corresponding with enhanced uptake of Met, Ile, His, and Arg (p < 0.05). The mammalian target of rapamycin (mTOR) signaling pathway was activated as evidenced by increased phosphorylation of P70 S6 kinase (P70S6K) and mTOR (p < 0.05), while the general control nonderepressible 2 (GCN2) pathway was suppressed through reduced eukaryotic initiation factor 2α (eIF2α) phosphorylation (p < 0.05). As Phe concentration increased, its net uptake increased linearly (P_linear < 0.05) while uptake efficiency decreased linearly (P_linear < 0.05). High Phe concentration (0.56 mM) inhibited amino acid transporter expression and reduced uptake of Leu, Pro, and Tyr (p < 0.05). Additionally, Phe-to-Tyr conversion was dynamically regulated, with phenylalanine hydroxylase (PAH) activity inhibited at 0.07 mM Phe (p < 0.05) but enhanced at higher concentrations, concurrent with reduced exogenous Tyr uptake (p &lt; 0.05). These findings show that casein synthesis in BMECs is optimal at 0.14 mM Phe, coinciding with enhanced expression of amino acid transporters and activation of protein synthesis pathways. In contrast, higher Phe concentrations (0.56 mM) are associated with reduced amino acid utilization efficiency. These observations suggest potential mechanisms by which Phe concentration may regulate milk protein synthesis in dairy cows.