Effect of protein level and methionine supplementation on dairy cows during the transition period.

Cows experience a significant negative protein balance during the first 30 d of lactation. Given the functional effects of AA on health, especially in challenging periods such as calving, higher levels of protein and specific AA in the diet may act to improve health and feed intake. The response of dairy cows to 3 protein supplementation strategies during the transition period and through the first 45 d in milk was evaluated. The final data set had 39 Holstein cows blocked based on parity (primiparous vs. multiparous) and expected calving and randomly assigned within each block to one of 3 dietary treatments: low protein (LP), high protein (HP), or high protein plus rumen-protected methionine (HPM). Treatments were offered from d -18 ± 5 to 45 d relative to parturition. Pre- and postpartum diets were formulated for high metabolizable protein (MP) supply from soybean meal, and HP and HPM provided higher MP balance than LP. Preplanned contrasts were LP versus HP+HPM and HP versus HPM. Significance was declared at P ≤ 0.05 and trends at 0.05

Lactation performance of dairy cows fed rehydrated and ensiled corn grain differing in particle size and proportion in the diet.

Rehydrated and ensiled mature ground corn has high ruminal starch digestibility, but particle size (PS) and dietary starch proportion (ST) can affect starch digestion and lactating cow performance. We evaluated the effect of rehydrated and ensiled corn (REC), PS, and ST on intake, lactation performance, nutrient digestibility, ruminal fermentation profile, and chewing behavior of dairy cows. Kernels from an 84% vitreousness hybrid were finely (FN) or coarsely (CS) ground, yielding geometric mean particle sizes of 1,591 and 2,185 µm, respectively. Ground kernels were rehydrated [60% dry matter (DM)] and ensiled in 200-L buckets for ≥205 d. The grinding rate (t/h) was 3.9 for FN and 11.7 for CS. The PS did not affect DM loss (11.3% of ensiled) or silage pH (3.8). Samples of each bucket (n = 15/PS) before and after silage fermentation were incubated in situ for 0, 3, 6, 18, and 48 h in 4 rumen-cannulated lactating cows. Ensiling increased the effective ruminal in situ DM degradation (63.7 vs. 34.1%), regardless of PS. Sixteen Holstein cows (152 ± 96 d in milk) in 4 × 4 Latin squares (21-d periods) were individually fed a 2 × 2 factorial combination of low (LO) or high (HI) starch diets with FN or CS. Cows were fed the same REC incubated in situ. Varied concentration of starch in the diet (29.2 vs. 23.5% of DM) was achieved by partial replacement of REC (22.0 vs. 14.2% of DM) with citrus pulp (0 vs. 8.2% of DM). Milk, protein, fat, and lactose yields did not differ. Milk fat percentage was reduced and protein percentage was increased by HI. Treatment FN increased feed efficiency (energy-corrected milk/digestible organic matter intake) when fed with HI. Total-tract starch digestibility tended to be reduced by CS (96.4 vs. 97.2% of starch intake). Serum ß-hydroxybutyrate was increased by LO. High-starch diet reduced the molar proportions of acetate and butyrate in ruminal fluid and increased propionate and isoacids. Particle size did not affect ruminal fermentation profile. Coarse grinding reduced plasma d-lactate concentration with HI. Diet HI reduced the proportion of daily intake from 1900 to 0700 h and induced preferential intake of feed particles <8 mm and greater refusal of particles >19 mm in the morning. Fine REC reduced rumination time per day and increased eating time per DM intake. Milk and plasma urea-N did not differ. Ensiling of mature flint corn for >200 d largely eliminated the effect of the PS of REC on the studied outcomes. The proportion of REC in the diet affected ruminal fermentation profile and milk solids concentration, but did not affect short-term performance and digestibility. Coarse grinding of REC may allow increasing the grinding rate and thus save labor and energy during ensiling.

Insulin signaling and antioxidant proteins in adipose tissue explants from dairy cows challenged with hydrogen peroxide are altered by supplementation of arginine or arginine plus methionine.

Arginine (Arg) and methionine (Met) can elicit anti-inflammatory and antioxidant effects in animals. Unlike Met, however, it is unknown if the supply of Arg can impact key aspects of adipose tissue (AT) function in dairy cows. Since Met and Arg metabolism are linked through the synthesis of polyamines, it is also possible that they have a complementary effect on aspects of AT function during a stress challenge. In this experiment, subcutaneous AT was harvested from four lactating multiparous Holstein cows (~27.0 kg milk per day, body condition score 3.38 ± 0.23) and used for incubations (4 h) with the following: control medium with an "ideal" profile of essential amino acids (IPAA; CTR; Lys:Met 2.9:1), IPAA plus 100 µM H2O2 (HP), H2O2 plus greater Arg supply (HPARG; Lys:Arg 1:1), or H2O2 plus greater Arg and methionine (Met) supply (HPARGMET; Lys:Met 2.5:1 and Lys:Arg 1:1). Western blotting was used to measure abundance of 18 protein targets associated with insulin and AA signaling, nutrient transport, inflammation, and antioxidant response. Reverse transcription polymerase chain reaction (RT-PCR) was used to assess effects on genes associated with Arg metabolism. Among the protein targets measured, although abundance of phosphorylated (p) AKT serine/threonine kinase (P = 0.05) and p-mechanistic target of rapamycin (P = 0.04) were lowest in HP explants, this effect was attenuated in HPARG and especially HPARGMET compared with CTR. Compared with HP, incubation with HPARG led to upregulation of the AA transporter solute carrier family 1 member 3 (L-glutamate transporter; P = 0.03), the reactive oxygen species detoxification-related enzyme glutathione S-transferase mu 1 (GSTM1; P = 0.03), and fatty acid synthase (P = 0.05). Those effects were accompanied by greater abundance of solute carrier family 2 member 4 (insulin-induced glucose transporter) in explants incubated with HPARG and also HPARGMET (P = 0.04). In addition, compared with other treatments, the peak response in abundance of the intracellular energy sensor 5'-prime-AMP-activated protein kinase was detected with HPARGMET (P = 0.003). There was no effect of Arg or Arg plus Met on the mRNA abundance of genes associated with Arg metabolism (ARG1, NOS2, AMD1, SMS, and SRM). Overall, supplementation of Arg alone or with Met partially alleviated the negative effects induced by H2O2. More systematic studies need to be conducted to explore the function of Arg supply with or without Met on AT function.

In nonruminants, oxygen-derived free-radicals such as hydrogen peroxide produced during stressful events impair insulin responsiveness including glucose uptake, protein synthesis, and fatty acid metabolism. Arginine and methionine supply induce anti-inflammatory and antioxidant responses during stressful conditions. We studied the acute effect of arginine supplementation alone or combined with methionine on protein abundance in adipose tissue explants from lactating Holstein cows challenged with hydrogen peroxide. Hydrogen peroxide reduced protein abundance of key insulin and amino acid signaling proteins. Most pronounced and positive effects were detected with arginine alone, restoring abundance of key target proteins including those involved in glucose, amino acid, and glutathione metabolism. Potential benefits of enhanced post-ruminal arginine supply during stressful periods such as the transition into lactation merit further study.

Branched-Chain Amino Acid Supplementation Alters the Abundance of Mechanistic Target of Rapamycin and Insulin Signaling Proteins in Subcutaneous Adipose Explants from Lactating Holstein Cows.

The objective of this study was to investigate changes in protein abundance of mTOR and insulin signaling pathway components along with amino acid (AA) transporters in bovine s.c. adipose (SAT) explants in response to increased supply of Leu, Ile, or Val. Explants of SAT from four lactating Holstein cows were incubated with high-glucose serum-free DMEM, to which the 10 essential AAs were added to create the following treatments: ideal mix of essential AA (IPAA; Lys:Met 2.9:1; Lys:Thr 1.8:1; Lys:His 2.38:1; Lys:Val 1.23:1; Lys:Ile 1.45:1; Lys:Leu 0.85:1; Lys:Arg 2.08:1) or IPAA supplemented with Ile, Val, or Leu to achieve a Lys:Ile of 1.29:1 (incIle), Lys:Val 1.12:1 (incVal), or Lys:Leu (incLeu) 0.78:1 for 4 h. Compared with IPAA, incLeu or incIle led to greater activation of protein kinase B (AKT; p-AKT/total AKT) and mTOR (p-mTOR/total mTOR). Total EAA in media averaged 7.8 ± 0.06 mmol/L across treatments. Incubation with incLeu, incIle, or incVal led to greater protein abundance of solute carrier family 38 member 1 (SLC38A1), a Gln transporter, and the BCAA catabolism enzyme branched-chain α-keto acid dehydrogenase kinase (BCKDK) compared with IPAA. Activation of eukaryotic elongation factor 2 (eEF2; p-eEF2/total eEF2) was also greater in response to incLeu, incIle, or incVal. Furthermore, compared with incLeu or incIle, incVal supplementation led to greater abundance of SLC38A1 and BCKDK. BCKDK is a rate-limiting enzyme regulating BCAA catabolism via inactivation and phosphorylation of the BCKD complex. Overall, data suggested that enhanced individual supplementation of BCAA activates mTOR and insulin signaling in SAT. Increased AA transport into tissue and lower BCAA catabolism could be part of the mechanism driving these responses. The potential practical applications for enhancing post-ruminal supply of BCAA via feeding in rumen-protected form support in vivo studies to ascertain the role of these AAs on adipose tissue biology.

Methionine and Arginine Supply Alters Abundance of Amino Acid, Insulin Signaling, and Glutathione Metabolism-Related Proteins in Bovine Subcutaneous Adipose Explants Challenged with N-Acetyl-d-sphingosine.

The objective was to perform a proof-of-principle study to evaluate the effects of methionine (Met) and arginine (Arg) supply on protein abundance of amino acid, insulin signaling, and glutathione metabolism-related proteins in subcutaneous adipose tissue (SAT) explants under ceramide (Ce) challenge. SAT from four lactating Holstein cows was incubated with one of the following media: ideal profile of amino acid as the control (IPAA; Lys:Met 2.9:1, Lys:Arg 2:1), increased Met (incMet; Lys:Met 2.5:1), increased Arg (incArg; Lys:Arg 1:1), or incMet plus incArg (Lys:Met 2.5:1 Lys:Arg 1:1) with or without 100 µM exogenous cell-permeable Ce (N-Acetyl-d-sphingosine). Ceramide stimulation downregulated the overall abundance of phosphorylated (p) protein kinase B (AKT), p-mechanistic target of rapamycin (mTOR), and p-eukaryotic elongation factor 2 (eEF2). Without Ce stimulation, increased Met, Arg, or Met + Arg resulted in lower p-mTOR. Compared with control SAT stimulated with Ce, increased Met, Arg, or Met + Arg resulted in greater activation of mTOR (p-mTOR/total mTOR) and AKT (p-AKT/total AKT), with a more pronounced response due to Arg. The greatest protein abundance of glutathione S-transferase Mu 1 (GSTM1) was detected in response to increased Met supply during Ce stimulation. Ceramide stimulation decreased the overall protein abundance of the Na-coupled neutral amino acid transporter SLC38A1 and branched-chain alpha-ketoacid dehydrogenase kinase (BCKDK). However, compared with controls, increased Met or Arg supply attenuated the downregulation of BCKDK induced by Ce. Circulating ceramides might affect amino acid, insulin signaling, and glutathione metabolism in dairy cow adipose tissue. Further in vivo studies are needed to confirm the role of rumen-protected amino acids in regulating bovine adipose function.

Comparação da bioequivalência entre duas formulações de montelucaste de sódio 10 mg comprimidos revestidos em voluntários sadios após a administração de dose única / Comparison of the bioequivalence between two formulations of montelukast sodium 10 mg coated tablets in healthy volunteers after a single dose administration

O estudo foi realizado para comparar a bioequivalência de duas formulações de montelucaste de sódio 10 mg comprimidos revestidos (Montelucaste de Sódio do Aché Laboratórios Farmacêuticos S/A., formulação teste e Singulairâ da Merck Sharp & Dohme Farmacêutica Ltda., formulação referência, Brasil) em 26 voluntários sadios. O estudo foi realizado através de um desenho aberto, randomizado, cruzado em dois períodos com tempo de washout de uma semana. As amostras de plasma foram obtidas ao longo de um intervalo de 24 horas. As concentrações de montelucaste foram determinadas através de um equipamento HPLC/MS/MS, utilizando loratadina como padrão interno. A partir dos dados de concentração plasmática obtidos individualmente, calcularam-se os seguintes parâmetros farmacocinéticos: ASC0-t, ASC0-¥ e Cmax. As médias para ASC0-t para as formulações teste e referência foram 2.733,63 ng.h/mL e 2.643,25 ng.h/mL, para ASC0-¥ foram 2.859,97 ng.h/mL e 2751,23 ng.h/mL e para Cmax foram 410,45 ng/mL e 421,04 ng/mL, respectivamente. As razões das médias geométricas foram de 101,88% para ASC0-t, 102,39% para ASC0-¥ e 96,50% para Cmax. Os intervalos com 90% de confiança foram 91,48 - 113,45% para ASC0-t, 92,17 - 113,74% para ASC0-¥ e 86,70 - 107,42% para Cmax. Uma vez que os intervalos de confiança de 90% para Cmax, ASC0-t e ASC0-¥ estiveram dentro da faixa de 80% a 125% proposta pelo Food and Drug Administration (FDA) e pela ANVISA (Agência Nacional de Vigilância Sanitária) conclui-se que o comprimido revestido de montelucaste de sódio de 10 mg é bioequivalente ao comprimido revestido de Singulairâ de 10 mg e, dessa forma, o produto teste pode ser considerado intercambiável na prática médica.