Milk urea nitrogen variation explained by differences in urea transport into the gastrointestinal tract in lactating dairy cows.

Milk urea nitrogen (MUN) and blood urea nitrogen are correlated with nitrogen balance and nitrogen excretion; however, there is also a genetic component to MUN concentrations that could be associated with differences in urea transport. It was hypothesized that a portion of the variation in MUN concentrations among cows is caused by variation in gastrointestinal and kidney urea clearance rates. Eight lactating cows with varying MUN concentrations while fed a common diet were infused with [15N15N]urea to determine urea N entry rate (UER), gastrointestinal entry rate, returned to ornithine cycle, urea N used for anabolism, urea N excretion in feces and urine. Urea clearance rates by the kidneys and gastrointestinal tract were calculated from isotopic enrichment of urea excretion in urine and gut entry rate, respectively, and plasma urea N concentrations (PUN). Over the course of the experiment, animals weighed an average of 506 ± 62 kg and produced 26.3 ± 4.39 kg of milk/d, with MUN concentrations ranging from 11.6 to 17.3 mg/dL (average of 14.9 ± 2.1 mg/dL). Plasma urea N was positively correlated with UER, urea N excretion in urine, and urea N used for anabolism. Plasma urea N and MUN were negatively correlated with gut clearance rates and ratio of gastrointestinal entry rate to UER. This relationship supports the hypothesis that differences in gut urea transport activity among animals causes variation in PUN and MUN concentrations, and that cows with high PUN and MUN are less efficient at recycling PUN to the gastrointestinal tract and thus may be more susceptible to ruminal N deficiencies when fed low RDP diets. Such biological variation in urea metabolism necessitates an adequate safety margin when setting regulations for maximal MUN levels as an indicator of herd N efficiency.

Effect of early grain feeding of beef steers on postabsorptive metabolism.

The objective of this study was to determine the effect of early weaning followed by a period of high-grain feeding on plasma acetate kinetics and signaling protein phosphorylation in LM tissue of growing steers. We hypothesized that early grain feeding would result in altered cell signaling and acetate use to support observed improvements in carcass gain and marbling. Fall-born Angus × Simmental steers were weaned at 106 ± 4 d of age (early weaned [EW]; n = 6) and fed a high-grain diet for 148 d or remained with their dams (normal weaned [NW]; n = 6) on pasture until weaning at 251 ± 5 d of age. Both treatments were subsequently combined and grazed on mixed summer pasture to 394 ± 5 d of age followed by a feedlot ration until harvest at 513 ± 5 d of age. Longissimus muscle tissue biopsies were collected at 253 ± 5 and 394 ± 5 d of age and at harvest. Total and phosphorylated forms of 5' adenosine monophosphate-activated protein kinase (AMPK) and downstream proteins of the mammalian target of rapamycin signaling pathway were determined by western blotting. Eight steers were used to assess acetate clearance at different age points via a bolus infusion of acetate (4 mmol/kg of BW). Early weaned steers had greater (P < 0.05) ADG than NW steers during the early grain feeding period. Phosphorylated to total ratios of ribosomal protein S6 (rpS6) and ribosomal protein S6 kinase 1 (S6K1) were significantly different during the early grain feeding period. Phosphorylated to total ratios of S6K1, rpS6, acetyl-CoA carboxylase, and 4E binding protein 1 and the absolute amount of phosphorylated AMPK were correlated with ADG, explaining 46% of the variance. Acetate clearance rates were less (P < 0.05) and synthesis rates were greater (P = 0.06) in EW steers during early grain feeding. Acetate synthesis rates were also greater (P < 0.05) in NW steers at harvest, suggesting a permanent shift in the gut microflora or gut function in response to the treatment. Neither treatment nor acetate infusion significantly affected plasma glucose or insulin concentrations. Plasma ß-hydroxybutyric acid concentrations increased with acetate infusion (P < 0.05). Based on these results, altered cell signaling during the early grain feeding period likely mediated increased protein deposition, leading to increased carcass weights, but observed changes in acetate appearance and clearance rates do not appear to explain the observed differences in intramuscular fat deposition during the terminal feeding period.

Acetate and glucose incorporation into subcutaneous, intramuscular, and visceral fat of finishing steers.

The objectives of this study were to assess the effects of early grain feeding on acetate and glucose turnover rates and acetate and glucose preference for palmitate synthesis by subcutaneous fat (SCF), intramuscular fat (IMF), and visceral fat (VF) in finishing steers. Sixteen Angus × Simmental steers were used in the study; 8 were early weaned (EW) and fed a high-grain diet immediately after weaning for 100 or 148 d, and 8 remained with their dams on pasture until weaning at 202 ± 5 or 253 ± 5 d of age. Normal weaned (NW) and EW animals were combined and grazed to 374 ± 5 or 393 ± 5 d of age, when they were placed on a corn silage-based finishing ration until they achieved a SCF thickness of 1.0 to 1.2 cm (494 ± 17 d of age for EW steers and 502 ± 12 d of age for NW steers). Immediately before harvest, steers were continuously infused for 12 h with [2H3] acetate (1.63 mmol/min; n = 8) or [U-13C6] glucose (0.07 mmol/min; n = 8). Blood samples were collected before initiation of infusions and at the end of the infusion from 8 animals or at 1-h intervals for the first 11 h and at 15-min intervals for the last hour of infusion for the other 8 animals. Adipose tissue samples from SCF, IMF, and VF depots were collected at harvest, and lipids were extracted. Plasma enrichments of acetate and glucose and palmitate enrichment in each depot were used to calculate plasma turnover rates and fractional synthesis rates (FSR; % per h) of palmitate from each isotope. Early weaned steers had greater marbling scores compared to NW steers ( P< 0.05). Plasma turnover rates and FSR for EW and NW steers were similar except for SCF, where a greater FSR from acetate was observed for EW steers. It is possible the greater FSR for SCF was due to harvesting the animals at a slightly more advanced stage of conditioning as evidenced by the trend for greater 12th rib fat (P = 0.07). Plasma acetate turnover and palmitate FSR from acetate were much greater (P < 0.05) than the corresponding rates from glucose, supporting the primary role of acetate as an energy source and the primary substrate for lipid synthesis across fat depots. However, FSR from acetate and glucose were not different among depots, suggesting that any potential effects of dietary starch on differential deposition of energy in SCF and IMF are not substrate driven.

Effects of AMP-activated protein kinase (AMPK) signaling and essential amino acids on mammalian target of rapamycin (mTOR) signaling and protein synthesis rates in mammary cells.

Regulation of mammary protein synthesis potentially changes the relationships between AA supply and milk protein output represented in current nutrient requirement models. Glucose and AA regulate muscle protein synthesis via cellular signaling pathways involving mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK). The objective of this study was to investigate the effects of essential AA (EAA) and acetate or glucose on mTOR and AMPK signaling pathways and milk protein synthesis rates. A bovine mammary epithelial cell line, MAC-T, was subjected to different media containing 0 or 3.5 mmol/L EAA concentrations with 0 or 5 mmol/L acetate or 0 or 17.5 mmol/L glucose in 2 separate 2 × 2 factorial studies. In a separate set of experiments, lactogenic bovine mammary tissue slices were subjected to the same treatments except that the low EAA treatment contained a low level of EAA (0.18 mmol/L). Supplementation of EAA enhanced phosphorylation of mTOR (Ser2448) and eukaryotic initiation factor 4E binding protein 1 (4EBP1, Thr37/46), and reduced phosphorylation of eukaryotic elongation factor 2 (eEF2, Thr56) in MAC-T cells. Concentration of ATP and phosphorylation of AMPK increased and decreased, respectively, in the presence of EAA in MAC-T cells. Acetate, EAA, or glucose numerically reduced AMPK phosphorylation by about 16% in mammary tissue slices. Provision of EAA increased phosphorylation of mTOR and 4EBP1, intracellular total EAA concentration, and casein synthesis rates in mammary tissue slices, irrespective of the presence of acetate or glucose in the medium. Phosphorylation of mTOR had a marginally negative association with AMPK phosphorylation, which was positively related to eEF2 phosphorylation. Casein synthesis rates were positively and more strongly linked to mTOR phosphorylation than the negative link between eEF2 phosphorylation and casein synthesis rates. A 100% increase in mTOR phosphorylation was associated with an increase in the casein synthesis rate of 0.74%·h(-1), whereas a 100% increase in eEF2 phosphorylation was related to a decline in the casein synthesis rate of 0.33%·h(-1). Although AMPK phosphorylation was responsive to cellular energy status and had a negative effect on mTOR-mediated signals in bovine mammary epithelial cells, its effect on milk protein synthesis rates appeared to be marginal compared with the mTOR-mediated regulation of milk protein synthesis by EAA.

Isoleucine and leucine independently regulate mTOR signaling and protein synthesis in MAC-T cells and bovine mammary tissue slices.

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.

Essential amino acids regulate both initiation and elongation of mRNA translation independent of insulin in MAC-T cells and bovine mammary tissue slices.

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.