Understanding the regulatory effects of individual amino acids (AA) on milk protein synthesis rates is important for improving protein and AA requirement models for lactation. The objective of this study was to examine the effects of individual essential AA (EAA) on cellular signaling and fractional protein synthesis rates (FSR) in bovine mammary cells. Omission of L-arginine, L-isoleucine, L-leucine, or all EAA reduced (P < 0.05) mammalian target of rapamycin (mTOR; Ser2448) and ribosomal protein S6 (rpS6; Ser235/236) phosphorylation in MAC-T cells. Phosphorylation of mTOR and rpS6 kinase 1 (S6K1; Thr389) decreased (P < 0.05) in the absence of L-isoleucine, L-leucine, or all EAA in lactogenic mammary tissue slices. Omission of L-tryptophan also reduced S6K1 phosphorylation (P = 0.01). Supplementation of L-leucine to media depleted of EAA increased mTOR and rpS6 and decreased eukaryotic elongation factor 2 (Thr56) phosphorylation (P < 0.05) in MAC-T cells. Supplementation of L-isoleucine increased mTOR, S6K1, and rpS6 phosphorylation (P < 0.05). No single EAA considerably affected eukaryotic initiation factor 2-α (eIF2α; Ser51) phosphorylation, but phosphorylation was reduced in response to provision of all EAA (P < 0.04). FSR declined when L-isoleucine (P = 0.01), L-leucine (P = 0.01), L-methionine (P = 0.02), or L-threonine (P = 0.07) was depleted in media and was positively correlated (R = 0.64, P < 0.01) with phosphorylation of mTOR and negatively correlated (R = -0.42, P = 0.01) with phosphorylation of eIF2α. Such regulation of protein synthesis will result in variable efficiency of transfer of absorbed EAA to milk protein and is incompatible with the assumption that a single nutrient limits protein synthesis that is encoded in current diet formulation strategies.

The objective of this study was to evaluate local molecular adaptations proposed to regulate protein synthesis in the mammary glands. It was hypothesized that AA and energy-yielding substrates independently regulate AA metabolism and protein synthesis in mammary glands by a combination of systemic and local mechanisms. Six primiparous mid-lactation Holstein cows with ruminal cannulas were randomly assigned to 4 treatment sequences in a replicated incomplete 4 x 4 Latin square design experiment. Treatments were abomasal infusions of casein and starch in a 2 x 2 factorial arrangement. All animals received the same basal diet (17.6% crude protein and 6.61 MJ of net energy for lactation/kg of DM) throughout the study. Cows were restricted to 70% of ad libitum intake and abomasally infused for 36 h with water, casein (0.86 kg/d), starch (2 kg/d), or a combination (2 kg/d starch+0.86 kg/d casein) using peristaltic pumps. Milk yields and composition were assessed throughout the study. Arterial and venous plasma samples were collected every 20 min during the last 8h of infusion to assess mammary uptake. Mammary biopsy samples were collected at the end of each infusion and assessed for the phosphorylation state of selected intracellular signaling molecules that regulate protein synthesis. Animals infused with casein had increased arterial concentrations of AA, increased mammary extraction of AA from plasma, either no change or a trend for reduced mammary AA clearance rates, and no change in milk protein yield. Animals infused with starch had increased milk and milk protein yields, increased mammary plasma flow, reduced arterial concentrations of AA, and increased mammary clearance rates and net uptake of some AA. Infusions of starch increased plasma concentrations of glucose, insulin, and insulin-like growth factor-I. Starch infusions increased phosphorylation of ribosomal protein S6 and endothelial nitric oxide synthase, consistent with changes in milk protein yields and plasma flow, respectively. Phosphorylation of the mammalian target of rapamycin was increased in response to starch only when casein was also infused. Thus, cell signaling molecules involved in the regulation of protein synthesis differentially responded to these nutritional stimuli. The hypothesized independent effects of casein and starch on animal metabolism and cell signaling were not observed, presumably because of the lack of a milk protein response to infused casein.

Current nutrient requirement models assume fixed efficiencies of absorbed amino acid (AA) conversion to milk protein. Regulation of mammary protein synthesis (PS) potentially violates this assumption by changing the relationship between AA supply and milk protein output. The objective of this study was to investigate the effects of essential AA (EAA) and insulin on cellular signaling and PS rates in bovine mammary cells. MAC-T cells were subjected to 0 or 100% of normal EAA concentrations in DMEM/F12 and 0 or 100 μg insulin/L in a 2 × 2 factorial arrangement of treatments. Lactogenic bovine mammary tissue slices (MTS) were subjected to the same treatments, except low-EAA was 5% of normal DMEM/F12 concentrations. In MAC-T cells, EAA increased phosphorylation of mammalian target of rapamycin (mTOR; Ser2448), ribosomal protein S6 kinase 1 (S6K1; Thr389), eIF4E binding protein 1 (4EBP1; Thr37/46), and insulin receptor substrate 1 (IRS1; Ser1101), and reduced phosphorylation of eukaryotic elongation factor 2 (eEF2; Thr56) and eukaryotic initiation factor (eIF) 2-α (Ser51). In the presence of insulin, phosphorylation of Akt (Ser473), mTOR, S6K1, 4EBP1, and IRS1 increased in MAC-T cells. In MTS, EAA had similar effects on phosphorylation of signaling proteins and increased mammary PS rates. Insulin did not affect MTS signaling, perhaps due to inadequate levels. Effects of EAA and insulin were independent and additive for mTOR signaling in MAC-T cells. EAA did not inhibit insulin stimulation of Akt phosphorylation. PS rates were strongly associated with phosphorylation of 4EBP1 and eEF2 in MTS. EAA availability affected translation initiation and elongation control points to more strongly regulate PS than insulin.

There are several models in the literature for predicting enteric methane (CH4 ) emissions. These models were often developed on region or country-specific data and may not be able to predict the emissions successfully in every region. The majority of extant models require dry matter intake (DMI) of individual animals, which is not routinely measured. The objectives of this study were to (i) evaluate performance of extant models in predicting enteric CH4 emissions from dairy cows in North America (NA), Europe (EU), and Australia and New Zealand (AUNZ) and (ii) explore the performance using estimated DMI. Forty extant models were challenged on 55, 105, and 52 enteric CH4 measurements (g per lactating cow per day) from NA, EU, and AUNZ, respectively. The models were ranked using root mean square prediction error as a percentage of the average observed value (RMSPE) and concordance correlation coefficient (CCC). A modified model of Nielsen et al. (Acta Agriculturae Scand Section A, 63, 2013 and 126) using DMI, and dietary digestible neutral detergent fiber and fatty acid contents as predictor variables, were ranked highest in NA (RMSPE = 13.1% and CCC = 0.78). The gross energy intake-based model of Yan et al. (Livestock Production Science, 64, 2000 and 253) and the updated IPCC Tier 2 model were ranked highest in EU (RMSPE = 11.0% and CCC = 0.66) and AUNZ (RMSPE = 15.6% and CCC = 0.75), respectively. DMI of cows in NA and EU was estimated satisfactorily with body weight and fat-corrected milk yield data (RMSPE < 12.0% and CCC > 0.60). Using estimated DMI, the Nielsen et al. (2013) (RMSPE = 12.7 and CCC = 0.79) and Yan et al. (2000) (RMSPE = 13.7 and CCC = 0.50) models still predicted emissions in respective regions well. Enteric CH4 emissions from dairy cows can be predicted successfully (i.e., RMSPE < 15%), if DMI can be estimated with reasonable accuracy (i.e., RMSPE < 10%).

In addition to lysine and methionine, current ration-balancing programs suggest that branched-chain amino acid (BCAA) supply may also be limiting in dairy cows. The objective of this study was to investigate whether BCAA, leucine, isoleucine, and valine become limiting for milk protein synthesis when methionine and lysine supply were not limiting. Nine multiparous Holstein cows with an average milk production of 53.5±7.1 kg/d were randomly assigned to 7-d continuous jugular infusions of saline (CTL), methionine and lysine (ML; 12 g and 21 g/d, respectively), or ML plus leucine, isoleucine, and valine (ML+BCAA; 35 g, 15 g, and 15 g/d, respectively) in a 3×3 Latin square design with 3 infusion periods separated by 7-d noninfusion periods. The basal diet consisted of 40% corn silage, 14% alfalfa hay, and a concentrate mix, and respectively supplied lysine, methionine, isoleucine, leucine, and valine as 6.1, 1.8, 4.7, 8.9, and 5.3% of metabolizable protein. Dry matter intake (23.9 kg/d), milk yield (52.8 kg/d), fat content (2.55%), fat yield (1.33 kg/d), lactose content (4.77%), lactose yield (2.51 kg/d), and milk protein efficiency (0.38) were similar across treatments. Protein yield and protein content were not significantly different between ML (1.52 kg/d and 2.88%, respectively) and ML+BCAA (1.51 kg/d and 2.83%, respectively), but they were significantly greater than that of CTL (1.39 kg/d and 2.71%). Cows that received ML+BCAA had less milk urea nitrogen content (10.9 mg/dL) compared with milk of CTL cows (12.4 mg/dL) and ML cows (11.8 mg/dL). Whereas high-producing cows responded positively to methionine and lysine supplementation, no apparent benefits of BCAA supplementation in milk protein synthesis were found. Infusion of BCAA may have stimulated synthesis of other body proteins, probably muscle proteins, as evidenced by decreased milk urea nitrogen.