Rumen-protected methionine supplementation during the transition period under artificially induced heat stress: Effects on cow-calf performance.

Dairy cows experiencing heat stress (HS) during the precalving portion of the transition period give birth to smaller calves and produce less milk and milk protein. Supplementation of rumen-protected methionine (RPM) has been shown to modulate protein, energy, and placenta metabolism, making it a potential candidate to ameliorate HS effects. We investigated the effects of supplementing RPM to transition cows under HS induced by electric heat blanket (EHB) on cow-calf performance. Six weeks before expected calving, 53 Holstein cows were housed in a tiestall barn and fed a control diet (CON, 2.2% Met of MP) or a CON diet supplemented with SmartamineM (MET, 2.6% Met of MP, Adisseo Inc., France). Four weeks precalving, all MET and half CON cows were fitted with an EHB. The other half of the CON cows were considered thermoneutral (TN), resulting in 3 treatments: CONTN (n = 19), CONHS (n = 17), and METHS (n = 17). Respiratory rate (RR), skin temperature (ST), and rectal temperature (RT) were measured thrice weekly and core body temperatures recorded biweekly. Postcalving BW and BCS were recorded weekly, and DMI was calculated and averaged weekly. Milk yield was recorded daily and milk components were analyzed every third DIM. Biweekly AA and weekly nonesterified fatty acids (NEFA), BHB, insulin, and glucose were measured from plasma. Calf birth weight and 24 h growth, thermoregulation, and hematology profile were measured and apparent efficiency of absorption (AEA) of immunoglobulins was calculated. Data were analyzed using the MIXED procedure of SAS with 2 preplanned orthogonal contrasts: CONTN versus the average of CONHS and METHS (C1) and CONHS versus METHS (C2). Relative to TN, EHB cows had increased RT during the postcalving weeks and increased RR and ST during the entire transition period. Body weight, BCS, DMI, and milk yield were not affected by the EHB or RPM. However, protein percentage and SNF were lower in CONHS, relative to METHS cows. At calving, METHS dams had higher glucose concentrations, relative to CONHS, and during the postcalving weeks, the EHB cows had lower NEFA concentrations than TN cows. Calf birthweight and AEA were reduced by HS, whereas RR was increased by HS. Calf withers height tended to be shorter and RT were lower in CONHS, compared with METHS heifers. Overall, RPM supplementation to transition cows reverts the negative effect of HS on blood glucose concentration at calving and milk protein percentage in the dams and increases wither height while decreasing RT in the calf.

Mechanisms by which feeding synthetic zeolite A and dietary cation-anion difference diets affect mineral metabolism in multiparous Holstein cows: Part I.

The periparturient period is characterized by the increased demand for calcium (Ca) in dairy cows. This has resulted in the use of several different prepartal nutritional strategies to prevent hypocalcemia postpartum. The objective of our study was to determine the effects of feeding synthetic zeolite A (XZ), a diet with negative dietary cation-anion difference (-DCAD), or a positive-DCAD diet (CON) during the close-up period on peripartal mineral dynamics and hormones involved in calcium metabolism. To this end, 121 multiparous Holstein cows, blocked by lactation number and expected due date, were enrolled at 254 d of gestation and randomly assigned to 1 of 3 prepartum diets: CON (+190 mEq/kg; n = 40), -DCAD (-65 mEq/kg; n = 41), or a diet supplemented with sodium aluminum silicate (XZ; +278 mEq/kg, fed at 3.3% DM, targeting 500 g/d; n = 40; Protekta Inc.). Blood, urine, and saliva samples were collected from enrollment until parturition, with data analyzed and presented beginning 14 d before parturition (d -14) until parturition (d 0), and on d 1, 2, 3, 6, 9, 12, 15, 18, 21, 35, and 49 postpartum, to assess mineral and hormone dynamics. Total fecal collections were performed in a subset of 8 cows per treatment group to assess fecal mineral loss. Data were analyzed as a randomized complete block design in SAS. Cows fed XZ and -DCAD had higher blood Ca concentrations compared with CON-fed cows, with XZ-fed cows exhibiting the highest blood Ca concentrations pre- and postpartum. Cows fed XZ had decreased blood and salivary phosphorus (P), increased fecal water-extractable phosphate, and the highest blood calcium concentrations pre- and postpartum. Parathyroid hormone was unaffected by diet but was increased at parturition in all treatments. Serotonin concentrations were increased in -DCAD and XZ cows compared with CON during the prepartum period. Our data indicate that the XZ group's improvement in blood Ca concentrations pre- and postpartum is most likely regulated by a dietary P restriction. Taken together, these data suggest that XZ and -DCAD diets improve postpartum calcium metabolism; however, they appear to work through different mechanisms.

Post-ruminal supplies of glucose and casein, but not acetate, stimulate milk protein synthesis in dairy cows through differential effects on mammary metabolism.

Amino acids and glucose have been shown to regulate protein synthesis in the mammary gland through their effects on cellular signaling pathways. Acetate might also have an effect on protein synthesis via the AMP-activated kinase signaling pathway, because it is the main energy source for the mammary secretory cell. Thus, the objective of this experiment was to evaluate the effects of casein and energy-yielding nutrients (acetate and glucose), and their combination, on performance and mammary metabolism. Six multiparous Holstein cows, averaging 49 kg of milk/d, were used in a 6 × 6 Latin square design with 14-d periods. Cows were fed to 100% National Research Council requirements for metabolizable protein (MP) and energy (ME) for 9 d, after which they were feed-restricted for 5 d to 85% of their individual ad libitum intake and then abomasally infused with 1 of 6 treatments. Treatments were acetate (A), glucose (G), each at 5% of ad libitum ME intake, casein (C) at 15% of ad libitum MP intake, A + C, G + C, or a saline solution (negative control). Casein infused alone increased milk protein yield numerically, with 25% recovery of the infused casein in milk protein. Glucose infused alone increased milk and milk protein yield and promoted the highest efficiency of nitrogen utilization (37%), with an efficiency of MP use for milk protein of 58%. We discovered no effect of treatment on mammary plasma flow, and the increase in milk protein yield with glucose infusion was brought about by greater mammary AA clearance rate. Infusion of casein and glucose together further increased milk protein yield in an additive fashion, and 47% of the infused casein was recovered in milk protein. Acetate infused alone had no effect on milk protein yield but increased milk fat yield numerically, suggesting that the greater amount of acetate taken up by the mammary gland was used for milk fat synthesis. Infusion of acetate and casein together yielded responses similar to those of casein alone. In conclusion, glucose has a major effect on stimulating milk protein synthesis, and the mammary gland has the ability to increase its supply of nutrients to match its synthetic capacity.

Development of a model describing regulation of casein synthesis by the mammalian target of rapamycin (mTOR) signaling pathway in response to insulin, amino acids, and acetate.

To improve dietary protein use efficiency in lactating cows, mammary protein synthesis responses to AA, energy substrates, and hormones must be better understood. These entities exert their effects through stimulation of mRNA translation via control of initiation and elongation rates at the cellular level. A central protein kinase of this phenomenon is the mammalian target of rapamycin (mTOR), which transfers the nutritional and hormonal stimuli onto a series of proteins downstream through a cascade of phosphorylation reactions that ultimately affect protein synthesis. The objective of this work was to further develop an existing mechanistic model of mTOR phosphorylation responses to insulin and total essential AA to include the effects of specific essential AA and acetate mediated by signaling proteins including protein kinase B (Akt), adenosine monophosphate activated protein kinase (AMPK), and mTOR and to add a representation of milk protein synthesis. Data from 6 experiments in MAC-T cells and mammary tissue slices previously conducted in our laboratory were assembled and used to parameterize the dynamic system of differential equations representing Akt, AMPK, and mTOR in their phosphorylated and dephosphorylated states and the resulting regulation of milk protein synthesis. The model predicted phosphorylated Akt, mTOR, AMPK, and casein synthesis rates with root mean square prediction errors of 16.8, 28.4, 33.0, and 54.9%, respectively. All other dependent variables were free of mean and slope bias, indicating an adequate representation of the data. Whereas mTOR was not very sensitive to changes in insulin or acetate levels, it was highly sensitive to leucine and isoleucine, and this signal appeared to be effectively transduced to casein synthesis. Although prior work had observed a relationship with additional essential AA, and data supporting those conclusions were present in the data set, we were unable to derive significant relationships with any essential AA other than leucine and isoleucine. The signaling properties and dynamics of AMPK under nutrient depletion and sufficiency, the responses to additional essential AA, and the consequent effects on protein synthesis remain to be better understood.

Effects of reduced dietary protein and supplemental rumen-protected essential amino acids on the nitrogen efficiency of dairy cows.

When fed to meet the metabolizable protein requirements of the National Research Council, dairy cows consume an excess of N, resulting in approximately 75% of dietary N being lost to the environment as urine and feces. Reductions in environmental N release could be attained through an improvement in N efficiency. The objective of this study was to determine if the predicted reduction in milk yield associated with feeding a low-protein diet to lactating dairy cows could be avoided by dietary supplementation with 1 or more ruminally protected (RP) AA. Fourteen multiparous and 10 primiparous Holstein cows, and 24 multiparous Holstein × Jersey crossbred cows were used in a Youden square design consisting of 8 treatments and 3 periods. The 8 dietary treatments were (1) a standard diet containing 17% crude protein [CP; positive control (PC)], (2) a 15% CP diet [negative control (NC)], (3) NC plus RP Met (+M), (4) NC plus RP Lys (+K), (5) NC plus RP Leu (+L), (6) NC plus RP Met and Lys (+MK), (7) NC plus RP Met and Leu (+ML), and (8) NC plus RP Met, Lys, and Leu (+MKL). Dry matter intake was not affected by treatment. Crude protein intake was lower for NC and RP AA treatments compared with the PC treatment. No detrimental effect was detected of the low-CP diet alone or in combination with AA supplementation on milk and fat yield. However, milk protein yield decreased for NC and +MKL diets, and lactose yield decreased for the +MKL compared with the PC diet. Milk urea N concentrations were lower for all diets, suggesting that greater N efficiency was achieved by feeding the low-protein diet. Minimal effects of treatments on arterial plasma essential AA concentrations were detected, with only Ile and Val being significantly lower in the NC than in the PC diet. Phosphorylation ratios of signaling proteins known to regulate mRNA translation were not affected by treatments. This study highlights the limitations of requirement models aggregated at the protein level and the use of fixed postabsorptive efficiency to calculate milk protein requirements. Milk protein synthesis regulation by signaling pathways in vivo is still poorly understood.

Invited review: Current representation and future trends of predicting amino acid utilization in the lactating dairy cow.

In current dairy production systems, an average of 25% of dietary N is captured in milk, with the remainder being excreted in urine and feces. About 60% of total N losses occur postabsorption. Splanchnic tissues extract a fixed proportion of total inflow of each essential AA (EAA). Those EAA removed by splanchnic tissues and not incorporated into protein are subjected to catabolism, with the resulting N converted to urea. Splanchnic affinity varies among individual EAA, from several fold lower than mammary glands' affinity for the branched-chain AA to similar or higher affinity for Phe, Met, His, and Arg. On average, 85% of absorbed EAA appear in peripheral circulation, indicating that first-pass removal is not the main source of loss. Essential AA in excess of the needs of the mammary glands return to general circulation. High splanchnic blood flow dictates that a large proportion of EAA that return to general circulation flow through splanchnic tissues. In association with this constant recycling, EAA are removed and catabolized by splanchnic tissues. This results in splanchnic catabolism equaling or surpassing the use of many EAA for milk protein synthesis. Recent studies have demonstrated that EAA, energy substrates, and hormones activate signaling pathways that in turn regulate local blood flow, tissue extraction of EAA, and rates of milk protein synthesis. These recent findings would allow manipulation of dairy diets to maximize mammary uptake of EAA and reduce catabolism by splanchnic tissues. Dairy cattle nutrient requirement systems consider EAA requirements in aggregate as metabolizable protein (MP) and assume a fixed efficiency of MP use for milk protein. Lysine and Met sufficiency is only considered after MP requirements have been met. By doing so, requirement systems limit the scope of diet manipulation to achieve improved gross N efficiency. Therefore, this review focuses on understanding the dynamics of EAA metabolism in mammary and splanchnic tissues that would lead to improved requirement prediction systems. Inclusion of variable individual EAA efficiencies derived from splanchnic and mammary responses to nutrient and hormonal signals should help reduce dietary protein levels. Supplementing reduced crude protein diets with individual EAA should increase gross N efficiency to more than 30%, reducing N excretion by the US dairy industry by 92,000 t annually.

Casein synthesis is independently and additively related to individual essential amino acid supply.

Specific AA affect rates of milk protein synthesis in the mammary glands of lactating cows. The objective of this study was to quantify the rate of αS1-casein synthesis in response to Ile, Leu, Met, and Thr supplementation, and to test the single-limiting AA theory for milk protein synthesis by exploring interactions among these AA. Effects of Ile, Leu, Met, and Thr were studied in vitro with a composite design containing a central point repeated 4 times, with 2 axial points per AA and a complete 2(4) factorial. Other AA were at the concentration in Dulbecco's modified Eagle medium/F12 medium (DMEM). The experiment was replicated with mammary tissue from 5 lactating cows. Mammary tissue slices (0.12 ± 0.02 g) were incubated for 4h at 37°C in 5 mL of treatment medium containing (2)H5-Phe. Caseins were precipitated from cell homogenate supernatants. Enrichment with (2)H5-Phe of the N[34]LLRFFVAPFPE αS1 peptide was determined by matrix-assisted laser desorption/ionization-tandem time-of-flight (MALDI-TOF-TOF), which was used to determine enrichment of Phe in the transfer (t)RNA pool and αS1-casein fractional synthesis rates (CFSR). Data were analyzed with a polynomial mixed model containing linear, quadratic, and 2-factor interactions for Ile, Leu, Met, and Thr, and cow and residual as random factors. Interactions were not significant at P<0.1 and were removed from the model. Increasing concentrations of Ile, Leu, Met, and Thr simultaneously increased CFSR curvilinearly with a predicted maximum response of 4.32 ± 0.84%/h at 63% of DMEM concentrations. The maximum response to each of the 4 AA was at 71, 49, 60, and 32% of the concentration in DMEM, for Ile, Leu, Met, and Thr, respectively. These values correspond to 270, 120, 440, and 140% the plasma concentrations of Ile, Leu, Met, and Thr observed in lactating cows fed to meet National Research Council requirements, respectively. The CFSR estimated at those maxima were similar among AA (3.6 ± 0.6%/h). Individual AA effects on CFSR did not correlate with mammalian target of rapamycin (mTOR) signaling. Independent responses of CFSR to individual essential AA observed in this study contradict the single-limiting AA theory assumed in current requirement systems. The saturable responses in CFSR to these 4 AA also highlight the inadequacy of using a fixed postabsorptive AA efficiency approach for determining AA requirements for milk protein synthesis.

Isoleucine, leucine, methionine, and threonine effects on mammalian target of rapamycin signaling in mammary tissue.

Improved representation of postabsorptive N metabolism in lactating dairy cows requires a better understanding of protein synthesis regulation in the mammary glands. This study aimed to determine the quantitative effects of Ile, Leu, Met, and Thr on the phosphorylation state of signaling proteins that regulate protein synthesis. The experiment used a composite design with a central point, 2 axial points per AA, and a complete 2(4) factorial. All of the other AA were provided at the concentrations in Dulbecco's modified Eagle's medium. The experiment was replicated with tissues from 5 lactating cows. Mammary tissue slices (0.12 ± 0.02 g) were incubated for 4h. Total and site-specific phosphorylated mammalian target of rapamycin (mTOR; Ser2448), eukaryotic elongation factor (eEF) 2 (Thr56), ribosomal protein S6 (Ser235/236), and eukaryotic initiation factor 2α (Ser51) were determined by western immunoblotting. Tissue concentrations of the 4 AA studied responded linearly to media supply. Addition of Ile, Leu, Met, or Thr had no effect on eukaryotic initiation factor 2α phosphorylation. Isoleucine and Thr positively affected mTOR phosphorylation. However, the 2 AA had an antagonistic relationship. Similarly, Ile linearly increased ribosomal protein S6 phosphorylation, and Thr inhibited the Ile effect. In addition, eEF2 phosphorylation was linearly decreased by Ile and Leu. Threonine curvilinearly decreased eEF2 phosphorylation, Ile and Leu negatively interacted on eEF2, and Thr tended to inhibit Leu effects on eEF2. This work demonstrated saturable responses and interactions between AA on activation of the mTOR pathway. Incorporation of these concepts into milk protein response models will help to improve milk and milk protein yield predictions and increase postabsorptive N efficiency and reduce N excretion by dairy cows.