Accurate assessment of the nutritional content of feed ingredients is required for precise diet formulation. Characterizing ingredients in terms of absorption and digestibility of individual AA is challenging, and this information often relies on indirect methods. The purpose of this research was to evaluate an in vivo stable isotope-based method of determining plasma entry rates of individual AA from feather meal (FM), blood meal (BM), and a rumen-protected AA (RPMet). Abomasal infusions of unprotected Ile, Leu, Met, and sodium caseinate were used as control treatments to assess technique reliability and accuracy. Isotopic enrichment of plasma AA in response to a 2-h constant jugular infusion of a mixture of C labeled AA was measured and modeled using a dynamic 4-pool model, which was fitted to each AA by infusion to derive diet entry rates. The resulting entry rate matrix was used to derive plasma entry rates of individual AA from each ingredient by regression. The mean of plasma AA entry for abomasally infused Ile, Leu, and Met was 93.4 ± 7.35% of that infused, indicating that 6.6% was used by splanchnic tissues during first pass. The mean of the plasma essential AA entry for abomasally infused casein was 86.7 ± 4.81% of that present in the source protein, which represents a mean of 8.7% first-pass use assuming 95% digestibility. Individual AA appearances ranged from 86 to 93% of the source content except Ile, which was 73%. These fractional appearance percentages were similar to those previously reported when using a dietary regression approach. The mean plasma essential AA entry rate for FM was 52.7% of the AA in the source ingredient, with a range across AA of 48 to 58%. The mean plasma essential AA entry rate for BM was 47.5%, with a range of 30 to 61%. However, estimated Met availability from the RPMet was lower (9.9%) than expected (42%). This may be due to the relatively larger errors of measurement for Met entry rates and a small change in RPMet inclusion. Assuming that rumen-undegraded protein absorption is reflective of aggregated essential AA entry rates after correction for first-pass use, 52.6 and 61.2% of dietary FM and BM CP was absorbed from the intestine, respectively, which yielded an estimated intestinal digestibility of 70 and 66%, respectively. This method appears to provide an accurate and precise in vivo assessment of individual AA plasma entry rates that can be used to better characterize individual feed ingredients in ruminants. Such information will result in more robust economic assessments of feeds and increased precision of diet formulation.

Accurate estimates of mean nutrient composition of feeds, nutrient variance (i.e., standard deviation), and covariance (i.e., correlation) are needed to develop a more quantitative approach of formulating diets to reduce risk and optimize safety factors. Commercial feed-testing laboratories have large databases of composition values for many feeds, but because of potentially misidentified feeds or poorly defined feed names, these databases are possibly contaminated by incorrect results and could generate inaccurate statistics. The objectives of this research were to (1) design a procedure (also known as a mathematical filter) that generates accurate estimates of the first 2 moments [i.e., the mean and (co)variance] of the nutrient distributions for the largest subpopulation within a feed in the presence of outliers and multiple subpopulations, and (2) use the procedure to generate feed composition tables with accurate means, variances, and correlations. Feed composition data (>1,300,000 samples) were collected from 2 major US commercial laboratories. A combination of a univariate step and 2 multivariate steps (principal components analysis and cluster analysis) were used to filter the data. On average, 13.5% of the total samples of a particular feed population were removed, of which the multivariate steps removed the majority (66% of removed samples). For some feeds, inaccurate identification (e.g., corn gluten feed samples included in the corn gluten meal population) was a primary reason for outliers, whereas for other feeds, subpopulations of a broader population were identified (e.g., immature alfalfa silage within a broad population of alfalfa silage). Application of the procedure did not usually affect the mean concentration of nutrients but greatly reduced the standard deviation and often changed the correlation estimates among nutrients. More accurate estimates of the variation of feeds and how they tend to vary will improve the economic evaluation of feeds and risk assessment of diets, and provide the ability to implement stochastic programming.

Essential AA (EAA), particularly leucine, isoleucine, methionine, and histidine, possess signaling properties for promoting cellular anabolic metabolism, whereas methionine, lysine, and histidine are considered also to be substrate limiting AA. The objective of this study was to evaluate production responses to supplementation of 2 AA groups in a 2 × 2 factorial design. Eight cows (99 ± 18 days in milk) were assigned to 4 jugular infusion treatments consisting of saline (CON), methionine plus lysine plus histidine (MKH), isoleucine plus leucine (IL), or MKH plus IL, in a replicated 4 × 4 Latin square design. Periods were 18 d in length, comprising 8 d of rest followed by 10 d of jugular infusion. Daily infusion amounts were 21 g of methionine, 38 g of lysine, 20 g of histidine, 50 g of leucine, and 22 g of isoleucine. Cows were ad libitum fed a common diet consisting of 15.2% crude protein and 1.61 Mcal/kg NE L on a dry matter basis that was predicted to meet rumen degradable protein requirements but was 15% deficient in metabolizable protein. Milk and energy-corrected milk yields increased by 2.3 kg/d and 1.9 kg/d, respectively, with infused IL, and no change was observed for MKH. Milk protein concentration increased by 0.13 percentage units for MKH, whereas milk protein yield increased for both MKH and IL by 84 g/d and 64 g/d, respectively. The milk protein yield increase for MKH + IL was 145 g/d versus CON. Gross feed efficiency tended to increase with IL infusion, and N efficiency tended to increase with MKH infusion. Aggregate arterial EAA concentrations less Met, Lys, and His declined by 7.2% in response to MKH infusion. Arterial EAA less Ile and Leu also declined by 6.2% in response to IL infusion. Net total AA (TAA) and EAA uptake by the udder tended to increase in response to MKH infusion, whereas mammary blood flow increased in response to IL infusion, but TAA and EAA net uptakes were unaffected. Apparent udder affinity increased for TAA and EAA less Met, Lys, and His in response to MKH infusion, whereas affinity for EAA less Ile and Leu increased for IL infusion. Venous Met and Leu concentrations increased by 192% and 35% from the MKH and IL infusions, respectively, compared with CON, which indicates that intracellular concentration of these EAA changed substantially. Increases in milk protein yield were observed from 2 groups of amino acids independently and additively, which contradicts the single limiting amino acid theory that a single EAA will limit milk protein yield.

Extracellular amino acid profiles affect intracellular AA concentrations and profile as well as signaling proteins that regulate protein translation rates. The objective of this study was to assess whether various extracellular AA profiles and varied ratios of Lys to Met would increase the phosphorylation of signaling proteins related to protein metabolism. Six AA profiles, reflecting Dulbecco's modified Eagle's medium (DMEM), blood meal (BM), corn gluten meal (CGM), casein (CAS), plasma of lactating cows (PLA), and a negative control (NEG) represented the first factor (F1), and the ratio of Lys to Met (unaltered or set to 3:1) was the second factor (F2). Treatments were arranged in a 6 × 2 factorial manner, resulting in 12 treatments that were replicated 4 times. The total AA masses for all treatments were set to 659 mg/L (63% of DMEM) except NEG (0 mg/L). Confluent mammary epithelial cells were exposed to treatment media for 80 min (SD = 7.4). Intracellular concentrations of 17 AA were changed according to F1. The Met and Leu percent of total intracellular AA mass, as an example, varied from 0.58 (PLA) to 6.94 (NEG, +F2) for Met and 0.05 (NEG, −F2) to 4.63 (CGM, +F2) for Leu. Overall, balancing for Lys and Met at a 3:1 ratio increased intracellular concentrations of Lys and Met by 54 and 71%, respectively. Within the mechanistic target of rapamycin (mTOR) pathway, phosphorylation of mTOR (Ser2448), ribosomal protein S6 (Ser235/236), and eukaryotic initiation factor 4E binding protein 1 (Thr37/46) (4EBP1) were increased by all 5 AA profiles compared with the NEG control. We found no differences in phosphorylation state among the 5 AA profiles, indicating lack of sensitivity to various AA profiles. This lack of sensitivity between AA profiles might also be due to assay imprecision or other experimental limitations. Only phosphorylation of 4EBP1 was increased for F2. Phosphorylation of eukaryotic initiation factor 2 α subunit (Ser51) was unaffected by either F1 or F2 factors. Regression analyses indicated that intracellular concentrations of Met, Thr, Ile, and Leu predicted phosphorylation of mTOR-related proteins with adequate precision and accuracy, suggesting that multiple EAA dictate regulation, regardless of AA ratios. Changes in extracellular AA profiles translated to modified intracellular AA profiles, and no single profile uniquely stimulated phosphorylation of the mTOR pathway-related proteins.

No abstract

Within-farm variation in forage composition can be substantial and potentially costly, and it presents challenges for sampling the forage accurately. We hypothesized that day-to-day variation in forage neutral detergent fiber (FNDF) concentrations and diet variation caused by sampling error would have negative effects on production measures in lactating dairy cows. Twenty-four Holstein cows (73 d in milk) were used in 8 replicated 3 × 3 Latin squares with 21-d periods. Treatments were (1) control (CON), (2) variable (VAR), and (3) overreacting (ORR). On average, over the 21-d period, all 3 treatments were the same [24.7% FNDF and 48.2% forage dry matter (DM) composed of 67% alfalfa silage and 33% grass silage]. The CON treatment was essentially consistent day-to-day in total forage and FNDF concentrations and proportion of alfalfa and grass silages. The VAR treatment changed daily (in a random pattern) in proportion of alfalfa and grass silages fed, which resulted in day-to-day changes in FNDF (range was 21.5 to 28%). The ORR treatment varied in a 5-d cyclic pattern in total forage and FNDF concentrations (26, 24, 28, and 21.5% FNDF). Over the 21 d, ORR (25.1 kg/d) had higher DM intake compared with CON (24.5 kg/d) and VAR (24.3 kg/d). Milk production (42.8 kg/d), milk fat (3.5%), and milk protein (2.8%) were not affected by treatment; however, a treatment × day interaction was observed for milk production. Lower daily milk yields for VAR and ORR compared with CON were rare; they only followed sustained 4- and 5-d periods of feeding higher FNDF diets compared with CON. In contrast, increased daily milk yields for VAR and ORR versus CON were more frequent and followed sustained diet changes of only 2 or 3d. Lipolytic and lipogenic-related enzyme mRNA abundances in subcutaneous adipose tissue were not affected by treatment. Treatment × day interactions were observed for milk fatty acid markers of cellulolytic bacteria (iso-14:0, iso-15:0, iso-16:0) and lipolysis (18:0) and generally followed the expected response to changes in daily rations. Overall, extreme daily fluctuations in FNDF had no cumulative negative effect on production measures over a 21-d period, and daily responses to transient increases in FNDF were less than expected.

No abstract

Understanding uptake of AA by mammary tissue as supply varies is critical for predicting milk component production. Our objective was to develop an in vitro method to quantify cellular uptake, efflux, and intracellular metabolism of individual AA that could be implemented for evaluating these factors when AA supply and profile are varied. Bovine primary mammary epithelial cells were grown to confluency and exposed to medium with an AA profile and concentration similar to lactating dairy cow plasma for 24 h. Cells were then preloaded in medium enriched with 15 N-labeled AA for 24 h followed by removal of the 15 N-labeled medium and incubation with medium enriched with 13 C-labeled AA for 0, 15, 60, 300, 900, 1,800, and 3,600 s. Extracellular free AA and intracellular free and protein-bound AA were analyzed for concentrations and isotopic enrichment by gas chromatography-mass spectrometry. A dynamic, 12-pool model was constructed representing extracellular and intracellular free and protein-bound pools of an AA, and their respective 15 N and 13 C isotopes. Markov chain Monte Carlo simulation (n = 5,000) was conducted to evaluate prediction errors by deriving standard errors and posterior distributions for rate constants, fluxes, and pools. Cellular Ala influx and efflux were higher than Leu, reflecting Ala role in driving system L transport and the high capacity of sodium-dependent transport. The Ala and Leu turnover rates were 181 and 95, 580 and 857, and 74 and 157% per hour for extracellular, intracellular, and fast protein-bound pools, respectively. The intracellular and extracellular Ala to Leu ratios were quite different, meaning the blood AA profile is not the AA profile provided for protein translation. The high level of exchange and rapid turnover of pools provide a mechanism for matching the AA supplies to the precision necessary for translation. This also understates the importance of using experimental medium similar to what is observed in vivo given that some AA depend on other AA for influx (exchange driven). The average root mean squared prediction error across the isotope enrichments, pools, and concentrations was 9.7 and 14.1% for Ala and Leu, respectively, and collinearity among parameters was low, indicating adequate fit and identifiability. The described model provides insight on individual AA transport kinetics and a method for future evaluation of AA transport and intracellular metabolism when subjected to varying AA supplies.