Amino acids are not only precursors for but also signaling molecules regulating protein synthesis. Regulation of protein synthesis via AA occurs at least in part by alterations in the phosphorylation status of mammalian target of rapamycin (mTOR) pathway proteins. Although the ideal profile of Lys:Met to promote milk protein synthesis during established lactation in dairy cows has been proposed to be 3:1, aside from being the most-limiting AA for milk protein synthesis, the role of Met in other key biologic pathways such as methylation is not well characterized in the bovine. The objective of this study was to determine the influence of increasing supplemental Met, based on the ideal 3:1 ratio of Lys to Met, on intracellular metabolism related to protein synthesis and mTOR pathway phosphorylation status. MAC-T cells, an immortalized bovine mammary epithelial cell line, were incubated (n = 5 replicates/treatment) for 12 h with 3 incremental doses of Met while holding Lys concentration constant to achieve the following: Lys:Met 2.9:1 (ideal AA ratio; IPAA), Lys:Met 2.5:1 (LM2.5), and Lys:Met 2.0:1 (LM2.0). The ratios of Thr:Phe (1.05:1), Lys:Thr (1.8:1), Lys:His (2.38:1), and Lys:Val (1.23:1) were the same across the 3 treatments. Applying gas chromatography-mass spectrometry metabolomics revealed distinct clusters of differentially concentrated metabolites in response to Lys:Met. Lower Phe, branched-chain AA, and putrescine concentrations were observed with LM2.5 compared with IPAA. Apart from greater intracellular Met concentrations, further elevations in Met level (LM2.0) led to greater intracellular concentrations of nonessential AA (Pro, Glu, Gln, and Gly) compared with IPAA and greater essential AA (EAA; Met, Ile, and Leu) and nonessential AA (Pro, Gly, Ala, Gln, and Glu) compared with LM2.5. However, compared with IPAA, mRNA expression of β-casein and AA transporters (SLC7A5, SLC36A1, SLC38A2, SLC38A9, and SLC43A1) and mTOR phosphorylation were lower in response to LM2.5 and LM2.0. Overall, the results of this study provide evidence that increasing Met while Lys and the ratios of Phe, Thr, His, and Val relative to Lys were held constant could increase the concentration and utilization of intracellular EAA, in particular branched-chain AA, potentially through improving the activity of AA transporters partly controlled by mTOR signaling. Because EAA likely are metabolized by other tissues upon absorption, a question for future in vivo studies is whether formulating diets for optimal ratios of EAA in the metabolizable protein is sufficient to provide the desired levels of these AA to the mammary cells.

No abstract

BackgroundPregnancy and early life are critical periods of plasticity during which the fetus and neonate may be influenced by environmental factors such as nutrition. Maternal methionine (Met) supply in non-ruminants during pregnancy can affect offspring development and growth. Thus, the objective of this study was to investigate if increasing Met supply during late-pregnancy affects developmental parameters of the calf at birth and if either maternal Met or colostrum from Met-fed cows alters calf growth. Calves born to Holstein cows individually-fed a basal control [CON; 1.47 Mcal/kg dry matter (DM) and 15.3% crude protein] diet with no added Met or CON plus ethylcellulose rumen-protected Met (MET; Mepron® at 0.09% of diet DM; Evonik Nutrition & Care GmbH, Germany) during the last 28 ± 2 d of pregnancy were used. A total of 39 calves were in CON (n = 22 bulls, 17 heifers) and 42 in MET (n = 20 bulls, 22 heifers). At birth, calves were randomly allocated considering dam treatment and colostrum as follows: 1) calves from CON cows and colostrum from CON cows (n = 21); 2) calves from CON cows and colostrum from MET cows (n = 18); 3) calves from MET cows and colostrum from MET cows (n = 22); and 4) calves from MET cows and colostrum from CON cows (n = 20). All calves were housed, managed, and fed individually during the first 9 wk of life.ResultsDespite greater daily DM intake pre-partum in cows fed MET (15.7 vs. 14.4 ± 0.12 kg/d, P < 0.05), colostrum quality and quantity were not affected by maternal diet. At birth, MET calves had greater (P ≤ 0.05) body weight (BW, 44.1 vs. 42.1 ± 0.70 kg), hip height (HH, 81.3 vs. 79.6 ± 0.53 cm) and wither height (WH, 77.8 vs. 75.9 ± 0.47 cm). In contrast, concentrations of His, Lys, and Asn in plasma were lower (P ≤ 0.05) in MET calves. Regardless of colostrum source, the greater BW, HH, and WH in MET calves at birth persisted through 9 wk of age resulting in average responses of + 3.1 kg BW, + 1.9 cm HH, and + 1.8 cm WH compared with CON. Average daily gain during the 9 wk was (P < 0.05) 0.72 ± 0.02 kg/d in MET compared with 0.67 ± 0.02 kg/d in CON calves. Respiratory scores were normal and did not differ (P > 0.05) due to maternal Met supply or colostrum source. However, fecal scores tended to be lower (P ≤ 0.10) in MET calves regardless of colostrum source.ConclusionsIncreasing the maternal supply of MET during late-pregnancy enhanced growth in utero as well as during the pre-weaning and early post-weaning periods. Although the ~ 1 kg/d greater DM intake during the last 2–3 wk prior to parturition could explain a portion of the 2 kg extra body mass of MET calves at birth, other mechanisms potentially encompassing nutrient assimilation efficiency likely played a role. Assessing the exact mechanisms sensitive to supply of Met or total amino acid supply during the latter stages of growth in utero merit further research.Electronic supplementary materialThe online version of this article (10.1186/s40104-018-0298-1) contains supplementary material, which is available to authorized ...

Amino acids not only serve as precursors for protein synthesis but also function as signaling molecules that can regulate the mammalian target of rapamycin (mTOR) pathway. Methionine and Lys are the most-limiting AA for milk production and a ratio of ∼3:1 Lys:Met in the metabolizable protein has been determined to be ideal. Besides Met and Lys, recent studies have evaluated Ile, Leu, Val, and Thr as potentially limiting for milk protein synthesis. The objective of this experiment was to determine if varying the ratio of Lys:Thr, Lys:Ile, Lys:Val, and Lys:Leu while maintaining an ideal ratio of Lys:Met and fixed ratio of other essential AA (IPAA) elicits changes in intracellular metabolites, gene transcription related to protein synthesis, and phosphorylation status of mTOR pathway proteins. Immortalized bovine mammary epithelial cell line (MAC-T) cells were incubated for 12 h (n = 5 replicates/treatment) with IPAA (2.9:1 Lys:Met; 1.8:1 Lys:Thr; 2.38:1 Lys:His; 1.23:1 Lys:Val; 1.45:1 Lys:Ile; 0.85:1 Lys:Leu; 2.08:1 Lys:Arg) or IPAA supplemented with Thr, Ile, Val, and Leu to achieve a Lys:Thr 1.3:1 (LT1.3), Lys:Ile 1.29:1 (LI1.29), Lys:Val 1.12:1 (LV1.12), or Lys:Leu 0.78:1 (LL0.78). Compared with IPAA, metabolomics via gas chromatography-mass spectrometry revealed that increases in availability of Thr, Ile, Val, and Leu led to greater concentrations of essential AA (Leu, Ile, Thr), nonessential AA (Gly, Glu, Gln, Ser, Pro, Asp), and various metabolites including uric acid, phosphoric acid, N-acetylglutamic acid, and intermediates of glycolysis and the tricarboxylic acid cycle. Compared with other treatments, LV1.12 led to greater phosphorylation status of serine/threonine kinase B (Akt), mTORC1, and ribosomal protein S6 and lower phosphorylation of α subunit of eukaryotic translation initiation factor 2. In addition, LV1.12 upregulated abundance of CSN2 and both the abundance and promoter methylation of CSN1S1. Although LI1.29 led to the second highest response in mTORC1 phosphorylation status, it resulted in the lowest phosphorylation of Akt and eEF2 and mRNA abundance of CSN2 and various AA transporters (SLC7A5, SLC36A1, SLC38A2, SLC38A9, SLC43A1). Overall, data indicate that an increase in Val at an ideal ratio of Lys:Met could further enhance milk protein synthesis by altering intracellular concentrations of essential AA and metabolites that could play a regulatory role, increasing phosphorylation status of mTORC1 and key signaling proteins, and upregulation of AA transporters.

To our knowledge, most studies demonstrating the role of manipulating maternal nutrition on hindgut (i.e., large intestine) microbiota in the offspring have been performed in non-ruminants. Whether this phenomenon exists in cattle is largely unknown. Therefore, the objectives of the current study were to evaluate the impact of maternal post-ruminal supply of methionine during late-pregnancy in dairy cows on fecal microbiota and metabolome in neonatal calves, and their association with body development and growth performance during the preweaning period. To achieve this, heifer calves, i.e., neonatal female offspring, born to Holstein cows receiving either a control (CON) diet (n = 13) or CON plus rumen-protected methionine (MET; Evonik Nutrition & Care GmbH) during the last 28 days of pregnancy were used. Fecal samples from heifers were collected from birth until 6 weeks of age, i.e., the preweaning period. Fecal microbiota was analyzed with QIIME 2 whereas fecal metabolites were measured using an untargeted LC-MS approach. At birth, MET heifers had greater (P ≤ 0.05) BW, HH, and WH. During the preweaning period, no differences between groups were detected for starter intake (P = 0.77). However, MET heifers maintained greater (P ≤ 0.05) BW, HH and tended (P = 0.06) to have greater WH and average daily gain (ADG) (P = 0.10). Fecal microbiota and metabolome profiles through 42 days of age in MET heifers indicated greater capacity for hindgut production of endogenous antibiotics and enhanced hindgut functionality and health. Enhancing maternal post-ruminal supply of methionine during late-gestation in dairy cows has a positive effect on hindgut functionality and health in their offspring through alterations in the fecal microbiota and metabolome without affecting feed intake. Those alterations could limit pathogen colonization of the hindgut while providing essential nutrients to the neonate. Together, such responses contribute to the ability of young calves to achieve better rates of nutrient utilization for growth.