The effect of the content of lysine and methionine in metabolizable protein (MP) on lactation performance and N utilization in Chinese Holstein cows was determined. A control diet (C) was formulated to be adequate in energy but slightly limiting in MP. The concentration of Met and Lys in MP was 1.87 and 5.93%, respectively. The treatments were as follows (% of Met or Lys in MP): L=diet C supplemented with L-lysine-HCl at 0.49% on a dry matter (DM) basis (Met, 1.87; Lys, 7.00); M=diet C supplemented with 2-hydroxy-4-(methylthio)-butanoic acid (HMB) at 0.15% (Met, 2.35; Lys, 5.93); ML=diet C supplemented with 0.49% L-lysine HCl and 0.15% HMB (Met, 2.39; Lys, 7.10). The diets were fed to 60 Chinese Holsteins in mid-lactation (average days in milk=120, and milk yield=32.0 kg/d) for 8 wk. Milk yield was increased by supplementation of either Lys (1.5 kg/d) or Met (2.0 kg/d), and supplementation of both Lys and Met further increased milk yield (3.8 kg/d). There was no significant difference in dry matter intake across treatment groups. Cows on treatments M (3.95%) and ML (3.90%) had higher milk fat content than those on C (3.60%) and L (3.67%), but there were no significant differences in milk protein and lactose contents or somatic cell count among treatments. Supplementation of Met or Lys significantly increased Met or Lys concentration in arterial plasma. Treatment ML had a higher conversion of intake N to milk N and lower urea N concentrations in serum, urine, and milk than did treatment C. Supplementing HMB and L-lysine-HCl to provide approximately 2.3% Met and 7.0% Lys of the MP in diets slightly limiting in MP increased milk production, milk protein yield, and N utilization efficiency.

The objective of this study was to investigate effects of rumen-protected γ-aminobutyric acid (GABA) on dry matter intake, milk performance, and serum metabolites in Chinese Holstein lactating cows. Thirty-nine multiparous cows were blocked based on days in milk (60 ± 6.3 d; mean ± SD) and milk production (30.9 ± 4.17 kg; mean ± SD), and were randomly assigned to 1 of 4 treatments, with rumen-protected GABA added at levels of 0, 0.8, 1.6, or 2.4 g/d, the actual predicted available amounts being 0, 0.30, 0.61, or 0.91 g of GABA/d, respectively. The experiment lasted for 8 wk, with the first week for adaptation. Milk yield and milk compositions were recorded weekly, and serum concentrations of GABA, neuropeptide Y, and biochemical and antioxidant variables were analyzed in the first, fourth, and seventh weeks of the study. Dry matter intake linearly increased in cows receiving added GABA compared with that for the control. Addition of 0.8 g of GABA/d was associated with higher milk yield than the other treatments, but contents of milk protein and fat did not differ across the treatments. Dietary GABA tended to quadratically enhance the serum content of GABA (23.6, 30.2, 29.8, or 28.3 mmol/L for 0, 0.8, 1.6, or 2.4 g/d, respectively), and increased neuropeptide Y, with the highest value (3.07 ng/L) for 0.8 g of GABA/d. Nonesterified fatty acid quadratically decreased with GABA addition, with the lowest value (218.1 μmol/L) for 0.8 g of GABA/d. Serum glutathione peroxidase and superoxide dismutase quadratically increased in cows fed GABA, whereas serum malondialdehyde was quadratically reduced for all GABA groups. Rumen-protected GABA quadratically improved N efficiency across all treatments, contributing to the enhanced production of milk and milk protein and reduced N emission to the environment. In conclusion, addition of rumen-protected GABA is beneficial for early lactation dairy cows in terms of feed intake, lactation performance, and animal health.

Amino acids are the building blocks of proteins and serve as key molecular components upstream of the signaling pathways that regulate protein synthesis. The objective of this study was to systematically investigate the effect of essential AA ratios on milk protein synthesis in vitro and to elucidate some of the underlying mechanisms. Triplicate cultures of MAC-T cells and bovine mammary tissue explants (MTE) were incubated with the optimal AA ratio (OPAA; Lys:Met, 2.9:1; Thr:Phe, 1.05:1; Lys:Thr, 1.8:1; Lys:His, 2.38:1; and Lys:Val, 1.23:1) in the presence of rapamycin (control), OPAA, a Lys:Thr ratio of 2.1:1, a Lys:Thr ratio of 1.3:1, a Lys:His ratio of 3.05:1, or a Lys:Val ratio of 1.62:1 for 12 h; the other AA concentrations were equal to OPAA. In some experiments, the cells were cultured with OPAA with or without rapamycin (100 ng/mL) or with mammalian target of rapamycin (mTOR) small interference RNA, and the MTE were exposed to OPAA with rapamycin for β-casein expression. Among the treatments, the expression of β-casein was greatest in the MTE cultured with OPAA. In MAC-T cells, the OPAA upregulated the mRNA expression of SLC1A5 and SLC7A5 but downregulated the expression of IRS1, AKT3, EEF1A1, and EEF2 compared with the control. The OPAA had no effect on the mTOR phosphorylation status but increased the phosphorylation of S6K1 and RPS6. When the MTE were treated with rapamycin in the presence of OPAA, the expression of β-casein was markedly decreased. The phosphorylation of RPS6 and 4EBP1 also was reduced in MAC-T cells. A similar negative effect on the expression of RPS6KB1 and EIF4EBP1 was detected when the cells were cultured with either rapamycin or mTOR small interference RNA. The optimal AA ratio stimulated β-casein expression partly by enhancing the transport of AA into the cells, cross-talk with insulin signaling and a subsequent enhancement of mTOR signaling, or translation elongation in both MAC-T cells and bovine MTE.

Nuclear factor (erythroid-derived 2)-like factor 2 (Nrf2) is a transcription factor that binds to the antioxidant response element (ARE) in the upstream promoter region of many antioxidative genes. The Nrf2-ARE signaling plays a key role in the cellular antioxidant-defense system, but whether Nrf2 activation has protective effects against heat shock (HS) stress in mammary epithelial cells (MEC) remains unclear. The objective of this study was to determine whether tert-butylhydroquinone (tBHQ), a well-known Nrf2 activator, could attenuate heat stress-induced cell damage in MAC-T cells of the bovine MEC line. The MAC-T cells were exposed to HS (42.5°C for 1h) followed by recovery at 37°C to mimic HS. Compared with cells that were consistently cultured at normothermia (37°C), the cell viability levels significantly decreased after HS stress. In parallel, heat stress increased the reactive oxygen species levels and induced cellular apoptosis and endoplasmic reticulum stress. The MAC-T cells that were pretreated with tBHQ (10μM) for 2h followed by HS had a reduction in the loss of cell viability. The tBHQ pretreatment significantly decreased cellular reactive oxygen species levels and stress-related marker gene expression. The tBHQ-treated MAC-T cells showed strong Nrf2-ARE signaling activation and a nuclear accumulation of Nrf2 and upregulated expression of Nrf2-ARE downstream genes. Small interfering RNA silencing of Nrf2 in HS-treated MAC-T cells almost completely abolished the cytoprotective effects by tBHQ. Overall, our results demonstrated that HS could cause cell damage in cultured bovine MEC, and that activation of Nrf2 by tBHQ could attenuate HS-induced cell damage.