S. Lemosquet

Ingestion of a protein hydrolysate is accompanied by an accelerated in vivo digestion and absorption rate when compared with its intact protein

BACKGROUND It has been suggested that a protein hydrolysate, as opposed to its intact protein, is more easily digested and absorbed from the gut, which results in greater plasma amino acid availability and a greater muscle protein synthetic response. OBJECTIVE We aimed to compare dietary protein digestion and absorption kinetics and the subsequent muscle protein synthetic response to the ingestion of a single bolus of protein hydrolysate compared with its intact protein in vivo in humans. DESIGN Ten elderly men (mean +/- SEM age: 64 +/- 1 y) were randomly assigned to a crossover experiment that involved 2 treatments in which the subjects consumed a 35-g bolus of specifically produced L-[1-(13)C]phenylalanine-labeled intact casein (CAS) or hydrolyzed casein (CASH). Blood and muscle-tissue samples were collected to assess the appearance rate of dietary protein-derived phenylalanine in the circulation and subsequent muscle protein fractional synthetic rate over a 6-h postprandial period. RESULTS The mean (+/-SEM) exogenous phenylalanine appearance rate was 27 +/- 6% higher after ingestion of CASH than after ingestion of CAS (P < 0.001). Splanchnic extraction was significantly lower in CASH compared with CAS treatment (P < 0.01). Plasma amino acid concentrations increased to a greater extent (25-50%) after the ingestion of CASH than after the ingestion of CAS (P < 0.01). Muscle protein synthesis rates averaged 0.054 +/- 0.004% and 0.068 +/- 0.006%/h in the CAS and CASH treatments, respectively (P = 0.10). CONCLUSIONS Ingestion of a protein hydrolysate, as opposed to its intact protein, accelerates protein digestion and absorption from the gut, augments postprandial amino acid availability, and tends to increase the incorporation rate of dietary amino acids into skeletal muscle protein.

The production of intrinsically labeled milk protein provides a functional tool for human nutrition research

Oral or intravenous administration of labeled, free amino acids does not allow the direct assessment of protein digestion and absorption kinetics following dietary protein intake. Consequently, dietary protein sources with labeled amino acids incorporated within the protein are required. The aim of this study was to produce milk proteins intrinsically labeled with l-[1-(13)C]phenylalanine that would allow the assessment of protein digestion and absorption kinetics and the subsequent muscle protein synthetic response to dietary protein intake in vivo in humans. Two Holstein cows (body weight of 726 +/- 38 kg) were continuously infused with l-[1-(13)C]phenylalanine at 402 micromol/min for 44 to 48 h, during and after which plasma samples and milk were collected. After milk protein separation, casein was used in a subsequent human proof-of-principle experiment. Two healthy males (aged 61 +/- 1 yr; body mass index of 22.4 +/- 0.1 kg/m(2)) ingested 35 g of casein highly enriched with [1-(13)C] phenylalanine. Plasma samples were collected at regular intervals, and skeletal muscle biopsies were collected before and 6 h after casein ingestion. In the initial experiment, a total of 5.83 kg of l-[1-(13)C]phenylalanine-enriched milk protein (casein enrichment was 29.4 molar percent excess) was collected during stable isotope infusion in the cows. In the proof-of-principle study, ingestion of 35 g of intrinsically labeled casein resulted in peak plasma l-[1-(13)C]phenylalanine enrichments within 90 min after protein ingestion (9.75 +/- 1.47 molar percent excess). Skeletal muscle protein synthesis rates calculated over the entire 6-h period averaged 0.058 +/- 0.012%/h. The production of intrinsically labeled milk protein is feasible and provides dietary protein that can be used to investigate protein digestion and absorption and the subsequent muscle protein synthetic response in vivo in humans.

Whole-body glucose metabolism and mammary energetic nutrient metabolism in lactating dairy cows receiving digestive infusions of casein and propionic acid

This study analyzed the effect of propionate (C3) and casein (CN) on whole-body and mammary metabolism of energetic nutrients. Three multiparous Holstein cows fitted with both duodenal and ruminal cannulas were used in 2 replicated Youden squares with 14-d periods. Effects of CN (743 g/d in the duodenum) and C3 (1,042 g/d in the rumen) infusions, either separately or in combination as supplements to a grass silage diet, were tested in a factorial arrangement. The control diet provided 97% of energy and protein requirements. Within each period, blood samples were taken (d 11) from the carotid artery and the right mammary vein to determine net uptake of energetic nutrients. Plasma blood flow was calculated using the Fick principle (based on Phe and Tyr). On d 13, [6,6-(2)H(2)]glucose was infused in the jugular vein to determine whole-body glucose rate of appearance (Ra) based on enrichments in arterial plasma. Both C3 and CN treatments increased whole-body Ra (17% and 13%, respectively) but only CN increased milk (18%) and lactose (14%) yields, suggesting no direct link between whole-body Ra and milk yield. When CN was infused alone, the apparent ratio of conversion of CN carbon into glucose carbon was 0.31 but, when allowance was made for the CN required to support the extra milk protein output, the ratio increased to 0.40, closer to the theoretical ratio (0.48). This may relate to the observed increases in arterial glucagon concentrations for CN alone. Conversely, the apparent conversion of infused C3 carbon alone to glucose was low (0.31). With C3, mammary plasma flow increased as did uptakes of lactate, Ala, and Glu whereas the uptake for beta-hydroxybutyrate (BHBA) decreased. Mammary net carbon balance suggested an increase with C3 treatment in glucose, lactate, Ala, and Glu oxidation within the mammary gland. Mammary glucose uptake did not increase with CN treatment, despite an increase in glucose arteriovenous difference and extraction rate, because plasma flow decreased (-17%). Whereas CN, alone or in combination with C3, increased both lactose and protein yields, only mammary AA (and BHBA in CN alone) uptake increased because plasma flow decreased (-17%). These data suggest that the observed variations of milk lactose yield (and other milk components) are linked to metabolic interchanges between several energetic nutrients at both the whole-body and mammary levels and are not explained by increases in whole-body glucose availability.

Effects of glucose, propionic acid, and nonessential amino acids on glucose metabolism and milk yield in Holstein dairy cows

Whole-body glucose rate of appearance (Ra) responses and milk lactose secretion were compared in dairy cows receiving duodenal infusions of glucose (Glc), a mixture of 5 nonessential amino acids (NEAAm), or ruminal infusions of propionic acid (C3). Four mid-lactation Holstein cows, fitted with both duodenum and rumen cannulas, were used in a 4 x 4 Latin square design with 14-d periods. Cows were fed a grass silage-based diet (Ctrl) that provided 88% of net energy of lactation and 122% of protein requirements. Concentrate was formulated with wheat (21.5%) and barley (20%) containing some starch. Isoenergetic infusions (5.15 Mcal/d of digestible energy) of Glc into the duodenum (7.7 mol/d), C3 into the rumen (14.1 mol/d), or NEAAm into the duodenum (in mol/d; Ala: 1.60; Asp: 0.60; Glu: 5.94; Gly: 1.22; Ser: 2.45) were given as a supplement to the Ctrl diet. During each period on d 13, [6,6-(2)H(2)]glucose was infused into one jugular vein and blood samples were taken from the other jugular vein to measure glucose enrichment and determine Ra. Dry matter intake decreased slightly with the infusions (6%), but did not differ among them. Whole body glucose Ra averaged 502, 745, 600, and 576 mmol/h for Ctrl, Glc, C3, and NEAAm, respectively. It increased with the increase in energy supply (Ctrl vs. infusions) and differed according to the nutrients infused. The Ra response was higher with Glc and C3 than with NEAAm and higher with Glc than with C3. Plasma concentrations of insulin were not affected, but insulin-like growth factor 1 increased with infusions. Plasma glucagon increased with NEAAm, which could favor the increased Ra. Overall, milk lactose yield (137, 141, 142, and 130 mmol/h for Ctrl, Glc, C3, and NEAAm, respectively) was not modified by the infusions, but was lower with NEAAm compared with Glc and C3. Changes in lactose yield did not parallel the increase in Ra, and therefore the ratio of lactose yield to Ra decreased with the infusions and was lower in Glc compared with C3, suggesting a shift of glucose utilization away from lactose synthesis toward other pathways, including mammary metabolism. Intestinal Glc was the most efficient nutrient in terms of increasing glucose Ra; however, there was no direct link between the increases in whole body glucose Ra observed with the 3 types of nutrients and milk lactose yield.

Milk protein responses in dairy cows to changes in postruminal supplies of arginine, isoleucine, and valine.

An ideal profile of essential AA (EAA) can improve the efficiency of metabolizable protein (or PDIE, the equivalent in the INRA feeding system) utilization in dairy cows. Compared with other EAA, existing recommendations for the requirements of Arg, Ile, and Val are few and inconsistent. Four multiparous Holstein dairy cows at 22±6 wk of lactation received 4 treatments (duodenal infusions of 445±22.4 g/d of an EAA mixture complementing a low-protein diet in a 4×4 Latin square design with a period length of 1 wk). The control treatment provided a balanced supply (in % of PDIE) of 5.1% Arg, 5.2% Ile, and 5.9% Val, whereas in the 3 subsequent treatments of -Arg, -Ile, and -Val, the concentrations of these 3 EAA were reduced to 3.5, 4.1, and 4.5%, respectively. All treatments were made isonitrogenous and were balanced to provide 7 other EAA (Lys, Met, His, Leu, Phe, Thr, and Trp), according to the recommendations described in the literature. Combined, the diet and the infusions provided 14.3±0.1% crude protein on a dry matter basis, and 66.0±1.2 g of PDIE/Mcal of net energy for lactation. Neither dry matter intake (19.2 kg/d) nor milk yield (30.4±0.4 kg/d) was affected by treatments. The -Arg and -Ile treatments did not modify milk protein synthesis or the efficiency of N utilization. However, the -Val treatment decreased milk protein content by 4.9% and milk crude protein content by 4.3%, and tended to decrease the efficiency of N use for milk protein yield by 3.7% (compared with the control). These effects of Val were related to a decrease in the plasma concentration of Val as well as a trend toward decreasing plasma concentrations of Met, His, and the sum of all EAA and nonessential AA in the -Val treatment, which indicates a different utilization of all AA in response to the Val deficit. The deletion of Ile, compared with the deletion of Val, tended to decrease the milk protein-to-fat ratio by 3.8%. In conclusion, the supply of Arg at 3.5% of PDIE was not limiting for milk protein synthesis. The slight effect on the milk protein-to-fat ratio caused by decreasing the supply of Ile suggests a need to reevaluate the Ile requirement more precisely. A low Val supply could be limiting for milk protein synthesis, provided that the requirements of Lys, Met, and His are met.

Effect of amino acid or casein supply on whole-body, splanchnic, and mammary glucose kinetics in lactating dairy cows

This study was conducted to establish how AA supplied in a free form or as protein (casein, CN) affect the whole-body rate of appearance (WB Ra) of glucose, splanchnic and mammary glucose kinetics, and milk lactose secretion in lactating dairy cows. Five Holstein cows fitted with a rumen cannula and permanent indwelling catheters in the abomasum, portal, hepatic, and mesenteric veins, and one mesenteric artery, were used in a Youden square with 4 periods of 14 d each. Cows were fed a hay-based diet providing 100 and 70% of their net energy and metabolizable protein (MP) requirements, respectively. Treatments consisted of abomasal infusions of water (70% of MP requirements: control, Con), free AA (95% of MP requirements: AA1; and 120% of MP requirements: AA2), or CN (95% of MP requirements: CN1). The free AA mixture had the same profile as CN. On d 14 of each period, [6,6-(2)H(2)]glucose (25.8 mmol/h) was infused into a jugular vein, and blood samples (n=8) were taken over 4h from arterial, portal, hepatic, and mammary sources to measure glucose enrichment and concentration. Splanchnic and mammary plasma flows were determined by downstream dilution of para-aminohippurate and with the Fick principle, respectively. The last 6 milkings of each period were weighed and sampled to measure the yields of milk and components. The AA1 and CN1 treatments were not different for any of the measured parameters. Supplying AA linearly increased glucose WB Ra (AA2 vs. Con: +151 mmol/h) and liver net flux (+149 mmol/h). Utilization of glucose from the plasma compartment by the portal-drained viscera and liver and true portal absorption were not affected by AA supply. From these observations, we suggest that the increased WB Ra was due to increased net hepatic production. The AA from the infusion, in excess of that used to cover the increase in milk protein, were converted to glucose with an apparent efficiency close to 100% of maximum theoretical efficiency. Milk and lactose yields increased linearly with infusions of AA, by 14 and 16% with AA2 treatment, respectively. However, mammary glucose uptake was not significantly altered by AA infusions; this suggests that the mammary gland exerts active control on the uptake and utilization of glucose. For all treatments, the sum of true portal glucose absorption and true hepatic glucose production contributed more than 99% of WB Ra in the lactating cow; this would suggest that renal glucose synthesis makes only a small contribution to WB Ra under these conditions.

Dietary carbohydrate composition modifies the milk N efficiency in late lactation cows fed low crude protein diets.

Nitrogen emissions from dairy cows can be readily decreased by lowering the dietary CP concentration. The main objective of this work was to test whether the milk protein yield reduction associated with low N intakes could be partially compensated for by modifying the dietary carbohydrate composition (CHO). The effects of CHO on digestion, milk N efficiency (milk N/N intake; MNE) and animal performance were studied in four Jersey cows fed 100% or 80% of the recommended protein requirements using a 4×4 Latin square design. Four iso-energetic diets were formulated to two different CHO sources (starch diets with starch content of 34.3% and NDF at 32.5%, and fiber diets with starch content of 5.5% and NDF at 49.1%) and two CP levels (Low=12.0% and Normal=16.5%). The apparent digestible organic matter intake (DOMI) and the protein supply (protein digestible in the small intestine; PDIE) were similar between starch and fiber diets. As planned, microbial N flow (MNF) to the duodenum, estimated from the urinary purine derivatives (PD) excretion, was similar between Low and Normal CP diets. However, the MNF and the efficiency of microbial synthesis (g of microbial N/kg apparently DOMI) were higher for starch v. fiber diets. Milk and milk N fractions (CP, true protein, non-protein N (NPN)) yield were higher for starch compared with fiber diets and for Normal v. Low CP diets. Fecal N excretion was similar across dietary treatments. Despite a higher milk N ouput with starch v. fiber diets, the CHO modified neither the urinary N excretion nor the milk urea-N (MUN) concentration. The milk protein yield relative to both N and PDIE intakes was improved with starch compared with fiber diets. Concentrations of β-hydroxybutyrate, urea and Glu increased and those of glucose and Ala decreased in plasma of cows fed starch v. fiber diets. On the other hand, plasma concentration of albumin, urea, insulin and His increased in cows fed Normal compared with Low CP diets. This study showed that decreasing the dietary CP proportion from 16.5% to 12.0% increases and decreases considerably the MNE and the urinary N excretion, respectively. Moreover, present results show that at similar digestible OM and PDIE intakes, diets rich in starch improves the MNE and could partially compensate for the negative effects of Low CP diets on milk protein yield.

Diets rich in starch increase the posthepatic availability of amino acids in dairy cows fed diets at low and normal protein levels

Five mid-lactation multicatheterized Jersey cows were used in a 4×4 Latin square design to investigate whether the increase in milk N yield associated with diets rich in starch versus fiber could originate from changes in the splanchnic AA metabolism and if these changes depended upon the dietary crude protein (CP) content. Four isoenergetic diets were formulated to provide 2 different carbohydrate compositions [diets rich in starch (350g of starch and 310g of neutral detergent fiber/kg of dry matter) versus rich in fiber (45g of starch and 460g of neutral detergent fiber/kg of dry matter)] crossed by 2 different CP contents (12.0 vs. 16.5% CP). At the end of each treatment period, 6 hourly blood samples were collected from the portal and hepatic veins as well as the mesenteric artery to determine net nutrient fluxes across the portal-drained viscera (PDV), liver, and total splanchnic tissues. Dry matter and calculated energy intake as well as total absorbed energy were similar across treatments. However, the net portal appearance (NPA) of acetate, total volatile fatty acids, and β-hydroxybutyrate were higher with diets rich in fiber versus starch, whereas that of oxygen, glucose, butyrate, and insulin were lower. Concomitant to these changes, the percentage of N intake recovered as total AA (TAA) in the portal vein was lower for diets rich in fiber versus starch (42.3 vs. 51.4%, respectively), without, however, any difference observed in the NPA of the main AA used as energy fuels by the PDV (Glu, Gln, and Asp). Despite a higher NPA of TAA with starch versus fiber diets, no differences in the net hepatic flux of TAA, essential and nonessential AA were observed, resulting in a higher (+22%) net splanchnic release of AA and, hence, a greater (+7%) milk N yield. The net hepatic flux and hepatic fractional removal of none of the individual AA was affected as the main carbohydrate changed from fiber to starch, except for Gly and Lys, which were higher for the latter. After correcting for differences in NPA of TAA, the net hepatic uptake of TAA tended to be lower with starch versus fiber diets. The higher transfer of N from feed to milk with diets rich in starch is not the consequence of a direct sparing AA effect of glucogenic diets but rather the result of lower energy requirements by the PDV along with a higher microbial N flow to the duodenum. A better AA use by peripheral tissues with starch versus fiber diets was also hypothesized but more studies are warranted to clarify this issue.

Effects of soybean meal or canola meal on milk production and methane emissions in lactating dairy cows fed grass silage-based diets

This study evaluated the effects of soybean meal (SBM) and heat-moisture-treated canola meal (TCM) on milk production and methane emissions in dairy cows fed grass silage-based diets. Twenty-eight Swedish Red cows were used in a cyclic change-over experiment with 4 periods of 21 d and with treatments in 2 × 4 factorial arrangement (however, the control diet without supplementary protein was not fed in replicate). The diets were fed ad libitum as a total mixed ration containing 600 g/kg of grass silage and 400 g/kg of concentrates on a dry matter (DM) basis. The concentrate without supplementary protein consisted of crimped barley and premix (312 and 88 g/kg of DM), providing 130 g of dietary crude protein (CP)/kg of DM. The other 6 concentrates were formulated to provide 170, 210, or 250 g of CP/kg of DM by replacing crimped barley with incremental amounts of SBM (50, 100, or 150 g/kg of diet DM) or TCM (70, 140, or 210 g/kg of diet DM). Feed intake was not influenced by dietary CP concentration, but tended to be greater in cows fed TCM diets compared with SBM diets. Milk and milk protein yield increased linearly with dietary CP concentration, with greater responses in cows fed TCM diets compared with SBM diets. Apparent N efficiency (milk N/N intake) decreased linearly with increasing dietary CP concentration and was lower for cows fed SBM diets than cows fed TCM diets. Milk urea concentration increased linearly with increased dietary CP concentration, with greater effects in cows fed SBM diets than in cows fed TCM diets. Plasma concentrations of total AA and essential AA increased with increasing dietary CP concentration, but no differences were observed between the 2 protein sources. Plasma concentrations of Lys, Met, and His were similar for both dietary protein sources. Total methane emissions were not influenced by diet, but emissions per kilogram of DM intake decreased quadratically, with the lowest value observed in cows fed intermediate levels of protein supplementation. Methane emissions per kilogram of energy-corrected milk decreased more when dietary CP concentration increased in TCM diets compared with SBM diets. Overall, replacing SBM with TCM in total mixed rations based on grass silage had beneficial effects on milk production, N efficiency, and methane emissions across a wide range of dietary CP concentrations.

Changes in mammary metabolism in response to the provision of an ideal amino acid profile at 2 levels of metabolizable protein supply in dairy cows: Consequences on efficiency

The aim of this study was to compare the modifications in mammary gland metabolism by supplying an ideal versus an imbalanced essential AA (EAA) profile at low and high metabolizable protein (or PDIE, its equivalent in the INRA feeding system). Four lactating, multiparous Holstein cows received 4 treatments composed of 2 basal diets containing 2 levels of PDIE (LP or HP) and 2 different infusions of AA mixtures (AA- or AA+) in the duodenum. The AA+ mixture contained Lys, Met, Leu, His, Ile, Val, Phe, Arg, Trp, and Glu, whereas the AA- mixture contained Glu, Pro, and Ser. The infusion mixtures were iso-PDIE. The diet plus infusions provided 13.9 versus 15.8% of crude protein that corresponded to 102 versus 118g/kg of dry matter of PDIE in LP and HP treatments, respectively. The treatments were designed as a 2×2 crossover design of 2 levels of PDIE supply (LP vs. HP) with 28-d periods. Infusions of AA in the duodenum (AA- vs. AA+) were superimposed to diet within each 28-d period according to 2×2 crossover designs with 14-d subperiods. Increasing the PDIE supply tended to increase milk protein yield; however, the efficiency of PDIE utilization decreased and the plasma urea concentration increased, indicating a higher catabolism of AA. The AA+ treatments increased milk protein yield and content similarly at both levels of protein supply. This was explained by an increase in the mammary uptake of all EAA except His and Trp. The mammary uptake of non-EAA (NEAA) was altered to the increase in EAA uptake so that the total AA uptake was almost equal to milk protein output on a nitrogen basis. The ratio between NEAA to total AA uptake decreased from 46% in LPAA- to 40% in LPAA+, HPAA-, and HPAA+ treatments. The PDIE efficiency tended to increase in the AA+ versus the AA- treatments because the NEAA supply and the amount of NEAA not used by the mammary both decreased. Nevertheless, our AA+ treatments seemed not to be the ideal profile: the mammary uptake-to-output ratio for Thr was higher than 1 in LPAA-, but it decreased to 1 in all the other treatments, suggesting that Thr was deficient in these treatments. Conversely, an excess of His was indicated because its uptake was similar in AA+ and AA- treatments. In conclusion, balancing the EAA profile increased milk protein yield and metabolizable protein efficiency at both levels of protein supply by increasing the mammary uptake of EAA and altering the NEAA uptake, leading to less AA available for catabolism.