Valine and nonessential amino acids affect bidirectional transport rates of leucine and isoleucine in bovine mammary epithelial cells.

A more complete understanding of the mechanisms controlling AA transport in mammary glands of dairy cattle will help identify solutions to increase nitrogen feeding efficiency on farms. It was hypothesized that Ala, Gln, and Gly (NEAAG), which are actively transported into cells and exchanged for all branched-chain AA (BCAA), may stimulate transport of BCAA, and that Val may antagonize transport of the other BCAA due to transporter competition. Thus, we evaluated the effects of varying concentrations of NEAAG and Val on transport and metabolism of the BCAA Ala, Met, Phe, and Thr by bovine mammary epithelial cells. Primary cultures of bovine mammary epithelial cells were assigned to treatments of low (70% of mean in vivo plasma concentrations of lactating dairy cows) and high (200%) concentrations of Val and NEAAG (LVal and LNEAAG, HVal and HNEAAG, respectively) in a 2 × 2 factorial design. Cells were preloaded with treatment media containing [15N]-labeled AA for 24 h. The [15N]-labeled media were replaced with treatment media containing [13C]-labeled AA. Media and cells were harvested from plates at 0, 0.5, 1, 5, 15, 30, 60, and 240 min after application of the [13C]-labeled AA and assessed for [15N]- and [13C]-AA label concentrations. The data were used to derive transport, transamination, irreversible loss, and protein-synthesis fluxes. All Val fluxes, except synthesis of rapidly exchanging tissue protein, increased with the HVal treatment. Interestingly, the rapidly exchanging tissue protein, transamination, and irreversible-loss rate constants decreased with HVal, indicating that the significant flux increases were primarily driven by mass action with the cells resisting the flux increases by downregulating activity. However, the decreases could also reflect saturation of processes that would drive down the mass-action rate constants. This is supported by decreases in the same rate constants for Ile and Leu with HVal. This could be due to either competition for shared transamination and oxidation reactions or a reduction in enzymatic activity. Also, NEAAG did not affect Val fluxes, but influx and efflux rate constants increased for both Val and Leu with HNEAAG, indicating an activating substrate effect. Overall, AA transport rates generally responded concordantly with extracellular concentrations, indicating the transporters are not substrate-saturated within the in vivo range. However, BCAA transamination and oxidation enzymes may be approaching saturation within in vivo ranges. In addition, System L transport activity appeared to be stimulated by as much as 75% with high intracellular concentrations of Ala, Gln, and Gly. High concentrations of Val antagonized transport activity of Ile and Leu by 68% and 15%, respectively, indicating competitive inhibition, but this was only observable at HNEAAG concentrations. The exchange transporters of System L transport 8 of the essential AA that make up approximately 40% of milk protein, so better understanding this transporter is an important step for increased efficiency.

Effects of dietary starch and ruminally undegraded protein on glucogenic precursors in lactating dairy cows.

Gluconeogenesis is a large contributor to the blood supply of glucose carbons. The impact of varying dietary starch and ruminally degraded protein (RDP) on glucose entry, and the contributions of propionate and lactate to total plasma glucose entry were evaluated. Six cannulated, lactating, Holstein cows were fed one of four treatment diets arranged as a 2 × 2 factorial within a 4 × 4 partially replicated Latin Square design: (1) 8% RDP (LRDP) and 16% starch (LSt), (2) LRDP and 30% starch (HSt), (3) 11% RDP (HRDP) and LSt, or (4) HRDP and HSt. On d 12 of each period, 2-[13C]-sodium propionate (0.15 g/h) was ruminally infused for 4 h; on d 13, 1,2-[13C2]-glucose (0.2 g/h) was infused into the jugular vein for 1 h followed by 1-[13C]-lactate (0.1 g/h) for 1 h. Blood samples were serially collected starting prior to the infusions, and analyzed for plasma glucose, propionate, and lactate isotopic ratios. A one-compartment, glucose carbon model with inputs from lactate, propionate, and other glucogenic precursors (Oth, primarily absorbed glucose plus amino acids) was fitted to the isotope ratio data to derive glucose entry rates and conversion of the precursors to glucose. Milk protein production additively increased when HSt and HRDP were fed (P = 0.05 and P = 0.02, respectively). Plasma glucose and propionate concentrations increased with HSt (P = 0.04 and P = 0.01, respectively) and LRDP (P = 0.02 and P < 0.01, respectively). Total glucose and Oth entry increased (P = 0.03 and P = 0.03, respectively) with HSt, indicating greater glucose absorption from the small intestine or conversion of amino acids to glucose in the liver. However, neither entry rate was affected by RDP. The lack of an RDP effect suggests the increase in microbial outflow in response to RDP did not significantly alter glucose precursor supplies. Entry rates of propionate and lactate carbon to glucose carbon were not affected by treatment suggesting that neither starch nor RDP significantly affected fermentation or lactate production. Derivation of absolute entry rates and contributions to glucose using isotopic tracers is complicated by single carbon removals in the pentose phosphate (PPP), tri-carboxylic acid (TCA), and gluconeogenic pathways, and label randomization with the PPP and TCA pathways. Multiple tracers must be used to avoid assumptions regarding the proportional entries. These results provide insights on glucose supply and contributors, and draw attention to significant label cycling when utilizing isotope techniques.

Assessing availability of amino acids from various feedstuffs in dairy cattle using a stable isotope-based approach.

Nitrogen efficiency in dairy cows can be improved by more precisely supplying essential amino acids (EAA) relative to animal needs, which requires accurate estimates of the availability of individual EAA from feedstuffs. The objective of this study was to determine EAA availability for 7 feed ingredients. Seven heifers (258 ± 28 kg BW) were randomly chosen and assigned to 8 treatment sequences in a 7 × 8 incomplete Latin square design. Treatments were a basal diet (BD), and 10% (on a dry matter basis) of BD replaced by corn silage (CS), grass hay (GH), alfalfa hay (AH), dried distillers grain (DDGS), soybean hulls (SH), wet brewers grain (BG), or corn grain (CG). Total plasma AA entry rates were estimated for each EAA within each diet by fitting a 4-pool dynamic model to observed plasma, 13C AA enrichment resulting from a 2-h constant infusion of a 13C algal AA mixture. Individual EAA availability from each test ingredient was determined by regression of entry rates for that AA on crude protein intake for each ingredient. The derived plasma total EAA entry rates for corn silage, grass hay, alfalfa hay, dried distillers grain, soyhulls, brewers grain, and corn grain were 30.6 ± 3.4, 27.4 ± 3.2, 31.3 ± 3.4, 37.2 ± 3.2, 26.4 ± 3.2, 37.8 ± 3.2, and 33.5 ± 3.2% (±standard error) of EAA from each ingredient, respectively. Using the previous estimate of 8.27% EAA utilization by splanchnic tissues during first pass, total rumen-undegradable protein EAA absorbed from the gut lumen was 33.4, 29.9, 34.1, 40.6, 28.8, 41.2, and 36.5% of the EAA in each ingredient respectively.

An in vivo stable isotope-based approach for assessment of absorbed amino acids from individual feed ingredients within complete diets.

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 13C 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.