Effect of the methodology on circulating peptides determination and consequences on net flux measurements across the gastrointestinal tract of sheep.

Two methodologies for the measurement of peptide amino acids (PAA) in blood were compared to evaluate their effects on the measurement of the net flux of peptides across the gastrointestinal tract of sheep. These methods consisted of a chemical deproteinization of blood samples with sulfosalicylic acid (1.6 M, 0.1 ml for 1 ml of sample) or perchloric acid (1 M, 1 ml for 1 ml of sample) followed by ultrafiltration through a 3,000-Da cut-off filter (SSA + UF3 kD) or gel filtration through a Sephadex G-15 column (1,500-Da cut-off filter; PCA + G-15), respectively, prior to PAA analysis. Peptide concentrations as determined by amino acid concentrations before and after hydrolysis of samples were slightly greater with the SSA + UF3 kD (991 microM) than with the PCA + G-15 (605 microM) methodology. However, both methodologies gave similar net portal-drained viscera flux data in sheep fed on alfalfa pellets with histidine as the only significant uptake of peptide amino acid.

Milk protein response to abomasal or mesenteric vein essential amino acid infusion in lactating dairy cows.

Previous experiments performed at our location suggested that the milk protein response to infusions of mixed essential amino acids (EAA) was greater when these were supplied via intravenous rather than abomasal or duodenal routes. However, as far as we are aware there have been no direct comparisons of the milk protein response to site of EAA provision in the same animals. Our objective was to directly compare the milk protein responses when cows were given mixtures of EAA provided via abomasal or mesenteric vein infusions. Four multiparous, ruminally cannulated, multicatheterized Holstein x Friesian cows averaging 18 wk postpartum were fed dehydrated alfalfa, grass silage, and low protein (11.9% CP, DM basis) concentrates at 30, 20, and 50%, respectively, of dry matter (DM) offered. Total mixed rations (14% crude protein, DM basis) were fed daily as 3 equal meals at 8-h intervals and 95% of ad libitum DM intake. The experimental design was a single reversal with two 10-d infusion periods separated by a 10-d rest period. Each infusion period consisted of 4-d control infusions into the mesenteric vein (saline at 2 ml/min) and abomasum (water at 9 L/d) followed by 6-d infusion of a mixture of EAA equal to 400 g of milk protein/d into the abomasum or mesenteric vein. Control infusions continued into the site not receiving EAA. Intake of DM (20.3 kg/d), milk yield (28.9 kg/d), and milk fat concentration (41.2 g/kg) were not affected by EAA infusions. Milk protein concentration (33.4 vs 34.6 g/kg) and output (938 vs. 982 g/d) were increased and milk lactose concentration was decreased (46.6 vs. 46.1 g/kg) by EAA, but the responses were not affected by infusion site. Recovery of EAA as increased milk protein output (10.9%) was similar for intravascular and abomasally infused EAA in these cows, but these responses were low compared with published effects of postruminal casein infusion.

Vascular sources of amino acids for milk protein synthesis in goats at two stages of lactation.

An arteriovenous technique, combined with a 30-h i.v. infusion of [5-(13)CH3]Met and [5,5,5-(2)H]Leu, was used to monitor mammary uptake of free amino acid (AA) and to estimate the proportion of casein synthesized from circulating peptides in goats in early and late lactation. At both stages, kinetics was performed on the last day of consecutive 5.5-d periods. The first period was an i.v. infusion of saline and the second an i.v. infusion of lysine (8.9 g/h) plus methionine (2 g/h). Net uptake of essential AA and protein yields were higher in early than in late lactation. Uptake of free Met, His, and Pro was less than, uptake of Tyr and Lys was equal to, and uptake of Arg, Leu, Val, and Ile was greater than milk protein synthesis. Peptide uptake, estimated from the difference in casein and plasma free AA enrichment, accounted for a larger fraction of casein-Met (17 vs. 8%) and casein-Leu (27 vs. 12%) in late than in early lactation. Small decreases in mammary blood flow, AA transport activity, and AA concentrations accounted for the lower uptake of AA in late compared with early lactation. Based on our studies of several AA, the utilization of circulating peptides for casein synthesis appears to be a general phenomenon.

Amino acid exchange by the mammary gland of lactating goats when histidine limits milk production.

The aim of this study was to monitor amino acid (AA) exchange kinetics of the mammary gland in response to an imposed limitation on His supply for milk production. Lactating goats (n = 4, approximately 120 DIM) were fed a low protein ration that provided only 77% of metabolizable protein and 100% of energy requirements for milk production. The protein deficiency was alleviated by infusion into the abomasum of an AA mixture (67 g/d) including (+H; 4.4 g/d) or excluding (-H) His. Goats were assigned to treatments (6 to 7 d) according to a switchback design. On the last day of the first two periods, [U-13C]AA were continuously infused i.v. for 7 h and arterial and mammary vein blood was withdrawn to determine plasma AA concentration and enrichment. Flow probes monitored mammary blood flow. The secretion and enrichments of AA in milk casein were monitored each hour. A three-pool model of the gland was used to derive bi-directional rates of plasma AA exchange. Arterial plasma His concentration was lower during -H infusion (8 vs. 73 microM), but those of other AA changed little. Responses to low levels of plasma His were: 1) mammary blood flow increased by approximately 33%; 2) the gland's capacity to remove plasma His increased 43-fold, whereas the gland's capacity for other AA declined by two- to threefold; and 3) influx and efflux of His by the gland decreased. Thus, as the reduction in His efflux was insufficient to offset the reduced influx, milk protein yield decreased from 118 to 97 g/d.

Lysine metabolism by the mammary gland of lactating goats at two stages of lactation.

An arteriovenous kinetics technique was used to monitor mammary gland lysine and protein metabolism in goats (n = 4) at two stages of lactation (80 +/- 17 vs. 233 +/- 14 DIM) in response to an i.v. infusion of lysine (Lys) plus methionine (Met). At each stage of lactation [2-15N] and [1-13C; 6,6-2H2] Lys kinetics were performed on the last day of 5-d i.v. infusion of saline followed by Lys (370 mg/h) plus Met (84 mg/h, LM). Milk and protein yields and dry matter intake were higher in early than in late lactation, but LM infusion did not affect these variables. Regardless of stage of lactation, the absolute and fractional oxidation rates of Lys by the mammary gland increased in response to LM infusion. When corrected for Lys oxidation, net uptake of Lys by the gland was less than milk protein Lys secretion. However, correction for the contribution of peptides (15.8%) to Lys uptake brought net Lys uptake close into balance with milk Lys secretion. The present data suggests that when Lys is in excess of requirements, the mammary gland appears to dispose of the extra supply via the oxidative mechanism.

Subclinical infection with the nematode Trichostrongylus colubriformis increases gastrointestinal tract leucine metabolism and reduces availability of leucine for other tissues.

Gastrointestinal (GI) tract leucine metabolism was measured in 6- to 9-mo-old lambs subjected to trickle infection with Trichostrongylus colubriformis larvae and in separate animals that were not infected. Animals prepared with a jejunal catheter and with indwelling catheters into the aorta and the portal- (PDV) and mesenteric- (MDV) drained viscera were infused simultaneously with [1-13C] and [5,5,5-2H3] leucine to determine GI tract sequestration of leucine from arterial and luminal amino acid pools by tracer and tracee arteriovenous concentration differences. Leucine oxidative losses and net fluxes were also determined across the GI tract. Infection had no detectable effect on whole-body leucine flux, but it increased total GI tract leucine sequestration by 24% (P<.05) and GI tract oxidative losses of leucine by 22 to 41% (P<.01). Net PDV fluxes of leucine were decreased by 20 to 32% during the infection. The infection did not alter either the proportion of precursor leucine used by GI tract metabolism that was derived from the arterial leucine pool (.84 to .88) or the proportional sequestration of digesta-derived leucine during "first pass" absorptive metabolism (.12 to .18). These findings help to elucidate the metabolic basis for the reduced growth rates and nitrogen retention observed when animals are subjected to subclinical nematode infection.

Growth hormone and amino acid supply interact synergistically to control insulin-like growth factor-I production and gene expression in cultured ovine hepatocytes.

Many of the anabolic effects of growth hormone (GH) are indirect, occurring through GH-stimulated production of insulin-like growth factor-I (IGF-I) by the liver. As well as being regulated by GH, plasma IGF-I concentrations have been demonstrated to depend upon the level of dietary protein intake, with low protein diets being associated with reduced circulatory IGF-I levels. This inhibitory effect cannot be reversed by GH injection, suggesting that liver sensitivity to GH becomes impaired.To investigate the mechanisms through which protein supply affects GH sensitivity, primary cultures of ovine hepatocytes were grown in defined media, containing various proportions (0.2, 1.0 and 5.0) of jugular amino acid concentrations in fed sheep. Production of IGF-I by these cells was measured after 24 and 48 h in culture by radioimmunoassay. In the first 24-h period basal IGF-I production was the same in all defined media, and GH caused an approximately 2-fold increase in IGF-I release in cells grown in 1.0xor 5.0xamino acid media (P<0.01). Although GH appeared to increase IGF-I release in this period for cells grown in 0.2xamino acid media, this effect was not statistically significant. In the period from 24-48 h in defined media, both basal and GH-stimulated IGF-I production was dependent on amino acid availability (P<0.05 and P<0.001 respectively). Factorial analysis of variance demonstrated a strong positive interaction (P<0.001) between the effects of amino acid availability and GH, such that GH increased IGF-I production by more than 2-fold in cells grown in 5.0xamino acid media (P<0.01) but had no effect on production by cells grown in 1.0xor 0.2xamino acid media. Measurement of steady state concentrations of exon 1-derived IGF-I mRNAs using an RNase protection assay demonstrated that the observed effects on IGF-I peptide secretion were strongly associated with parallel effects at the RNA level. Incorporation of (35)S-methionine into cellular proteins over a 4-h period starting 20 h after transfer to defined culture media was not significantly reduced in 1.0xcompared with 5.0x amino acid media, although rates under both of these conditions were significantly higher than those seen in 0.2xamino acid media (P<0.01). The lack of correspondence between the dose-dependent effects of amino acid supply on cellular protein synthesis and those on basal and GH-stimulated IGF-I production, suggests that amino acid supply modulates IGF-I production through selective mechanisms. Steady state levels of the CCAAT/enhancer-binding protein beta (C/EBPbeta) isoforms, liver-enriched activating protein (LAP) and liver-enriched inhibitory protein (LIP) were determined by Western blotting. When levels of LAP were expressed relative to LIP levels in the same extracts, a significant decrease in the LAP:LIP ratio was observed in response to amino acid limitation (P<0.05). These data strengthen earlier arguments that synergistic interaction between the effects of amino acids and GH on hepatic IGF-I gene expression underlie nutrition-dependent changes in circulating IGF-I titres. The association between these effects and altered levels of C/EBPbeta isoforms suggests that CCAAT/enhancer mediated control of IGF-I gene expression may be involved in this phenomenon.

An isotope dilution model for partitioning leucine uptake by the liver of the lactating dairy cow.

An isotope dilution model for partitioning leucine uptake by the liver of the lactating dairy cow is constructed and solved in the steady state. If assumptions ae made, model solution permits calculation of the rate of leucine uptake from portal and hepatic arterial blood supply, leucine export into the hepatic vein, leucine oxidation and transamination, and synthesis and degradation of hepatic constitutive and export proteins. The model requires the measurement of plasma flow rate through the liver in combination with leucine concentrations and plateau isotopic enrichments in arterial, portal and hepatic plasma during a constant infusion of [1-13C]leucine tracer. The model can be applied to other amino acids with similar metabolic fates and will provide a means for assessing the impact of hepatic metabolism on amino acid availability to peripheral tissues. This is of particular importance when considering the dairy cow and the requirements of the mammary gland for milk protein synthesis.

Vascular sources of phenylalanine, tyrosine, lysine, and methionine for casein synthesis in lactating goats.

The contribution to casein biosynthesis of peptides derived from blood was examined in late lactation goats (254 to 295 d in milk). Ratios of mammary uptake of free amino acids (AA) in blood to output of AA in milk protein and ratios of the enrichments of Phe, Tyr, Met, and Lys at isotopic plateau in secreted milk casein to the free AA in arterial and mammary vein blood were monitored during the last 5 h of a 30-h continuous i.v. infusion of [1-13C]Phe, [2H4]Tyr, [5-13CH3]Met, and [2-15N]Lys on two occasions: before (control) and on d 6 of an i.v. infusion of Phe (6 g/d). During the control, uptakes of free Phe and Met were less than their output in milk. This result was comparable with the labeling kinetic results, suggesting that vascular peptides contributed 5 to 11% of Phe and 8 to 18% of Met. Free Tyr and Lys uptakes during the control were sufficient for milk output; however, the labeling kinetics indicated that 13 to 25% of the Tyr and 4 to 13% of the Lys were derived from peptides. Infusion of Phe increased the uptake of free AA but reduced the contribution of peptides toward Phe (0 to 3%) and Tyr (8 to 14%) supply for casein synthesis. Whole body hydroxylation of Phe to Tyr increased from 10 to 18% with the infusion of Phe; within the mammary gland, this conversion was lower (3 to 5%). Results suggest that the mammary utilization of peptides containing Phe and Tyr appears to depend on the supply of free AA in blood.

Uptake of acetylated peptides from the small intestine in sheep and their nutritive value in rats.

Acetylation is a potential method for protecting dietary peptides from degradation by rumen micro-organisms. As a first step in determining the nutritive value of acetylated peptides, their disappearance in the small intestine of sheep and their ability to support growth in a rat bioassay were measured. 15N-labelled peptides were prepared from lucerne which had been grown with 15N-labelled (NH4)2SO4 in the absence of Rhizobium. Peptides were prepared by enzymic hydrolysis of the extracted protein. Two peptide preparations were made using different proteinase mixtures. These mixtures contained peptides with an average molecular weight of 559 and 522 Da. They were treated with acetic anhydride, which resulted in 85 and 88% modification respectively, and their uptake from the small intestine was determined by injecting 1 g of untreated or acetylated peptides in a Cr-EDTA solution into the jejunum of two sheep fitted with jejunal catheters and ileal cannulas. Ileal digesta were collected and analysed for Cr and 15N. The uptake of dialanine (Ala2) and N-acetyl-Ala2 were compared in a similar way. The disappearance of 15N from lucerne peptides was high (88 and 93% respectively) and this was not affected significantly by acetylation (86 and 87%). Corresponding values for Ala2 and N-acetyl-Ala2 were both 96%, as measured by HPLC. It was therefore concluded that acetylation did not affect the uptake of peptides from the small intestine in sheep. Two feeding trials were carried out with rats. The first trial was carried out with a protein-free diet to which was added 10% lactalbumin or 5% lactalbumin and then a mixture of methionine-free amino acids, either alone or supplemented with Met, Gly-Met or acetylated Gly-Met. The rats grew equally well on all sources of Met, but failed to grow significantly on the mixture of Met-free amino acids. In the second trial the diet contained casein as 5.9% of the basal diet. Additional casein, pancreatic casein hydrolysate (peptides) and acetylated pancreatic casein hydrolysate (acetylated peptides) were compared as sources of amino acids, at inclusion rates of 100 g/kg final diet. Feed intake was similar with casein and peptides treatments, but was depressed by 23% with acetylated peptides. Live weight gain was 15 and 75% lower with the peptides and acetylated peptides diets respectively. Addition of lysine, arginine or histidine did not restore feed intake or weight gain of rats receiving acetylated peptides, but feed intake was restored immediately when peptides replaced acetylated peptides. When intake was restricted to 9 g/d and acetylated casein hydrolysate replaced half of the protein in the diet, rats gained weight less rapidly (1.44 v. 1.09 g/d) and retained less N, such that only 0.36 of the acetylated peptide-N was calculated to remain available to the animal. This N retention compared with 0.70 for unmodified casein. Thus, the rat bioassay indicated that certain specific peptides may well be of high nutritive value following acetylation, but that there may be problems of inappetance and inefficient utilization with acetylated peptide mixtures.

Application of a U-13C-labeled amino acid tracer in lactating dairy goats for simultaneous measurements of the flux of amino acids in plasma and the partition of amino acids to the mammary gland.

A preliminary study was conducted using lactating British Saanen goats (n = 5) at 109 to 213 d in milk that yielded 1.67 to 3.68 kg of milk/d to examine the application of a U-13C-labeled amino acid (AA) mixture obtained from hydrolyzed algal proteins as a tracer for measuring plasma flux (n = 5) and partition to the mammary gland (n = 3; arteriovenous difference) of 13 AA simultaneously. Except for Ile and Ser, there was incomplete (6 to 54%) equilibration of the tracer with AA from packed blood cells (> 90% erythrocytes) during the 6-h infusions. This result agreed with the large ratio of packed cells to gradients for plasma AA concentration that was also observed. However, net mass and isotope removals by the mammary gland were predominantly from plasma, indicating that the erythrocytes did not participate in kinetic exchanges. Plasma AA fluxes (millimoles per kilogram of metabolizable protein intake per kilogram of body weight 0.75) differed among goats that consumed different protein sources; however, overall rates were lowest for Met (5 to 14) and His (8 to 17) and highest for Leu (48 to 70) and Ala (53 to 88). On average, 25% of plasma flux was partitioned to the mammary gland. Less than 20% of His, Ser, Phe, and Ala were directed to the mammary gland; 20 to 30% of Arg, Thr, Tyr, and Leu were directed to the mammary gland; and 30 to 40% of Pro, Ile, Lys, and Val were directed to the mammary gland. The unidirectional AA flux in the mammary gland (AA apparently available for protein syntheses, oxidation, and metabolite formation) did not match the pattern that is required for casein synthesis, suggesting differences in the metabolic requirements of AA for nonmilk protein synthesis.

Absorption of amino acids from the intestine and their net flux across the mesenteric- and portal-drained viscera of lambs.

Experiments were conducted to compare the rates of apparent absorption (disappearance) of individual essential amino acids (EAA) from the small intestine with their net fluxes across the mesenteric- (MDV) and portal- (PDV) drained viscera in sheep given a pelleted alfalfa diet at two levels of intake. Disappearances of individual EAA across the region of the small intestine drained by the mesenteric arcade (jejunum to ileum) were similar to those across the whole of the small intestine (duodenum to ileum). The net MDV flux of each EAA was similar to its rate of disappearance, but, with the exception of threonine on the low intake level, the net PDV flux was lower (P < .05). Increasing the intake of alfalfa from 800 to 1,200 g/d increased the rate of disappearance of individual EAA between the duodenum and ileum by .56 (range .43 to .65) and between the jejunum and ileum by .51 (range .45 to .60). The MDV and PDV blood flows increased by .35 and .39, respectively, and, with the exception of valine, net fluxes of individual EAA increased by .39 (range .20 to .50) across the MDV and by .44 (range .21 to .71) across the PDV. When net fluxes across the MDV and PDV were measured simultaneously, the ratio of PDV: MDV flux for each EAA was less than (P < .05) unity (mean .61, range .55 to .69), even though all MDV blood enters the PDV, contributing approximately .45 of the total portal flow. This observation suggests that, in regions of the tract not drained by the MDV, extraction of arterial blood EAA for tissue and secretory protein synthesis must exceed the release of protein degradation products into the venous drainage. The results are discussed in terms of endogenous protein secretions into the lumen of the tract anterior to the small intestine.

Quantification of circulating peptides and assessment of peptide uptake across the gastrointestinal tract of sheep.

Gastrointestinal absorption of peptides was examined in sheep fed a forage-based diet. Peptide concentrations were determined in arterial, portal, and mesenteric blood and plasma by quantification of amino acid concentrations before and after acid hydrolysis of samples that had been first deproteinized then subjected to Sephadex G-15 gel-filtration to remove residual protein. In contrast to other studies of ruminants, peptide concentrations for individual amino acids were lower than for the corresponding free amino acids with peptide (expressed as a proportion of total nonprotein amino acid) representing not more than .25 to .3 of total amino acid. Peptide concentrations in arterial, mesenteric, and portal blood and plasma were similar, indicating that on this diet there was no net uptake of peptides from the small intestine (mesenteric-drained viscera, MDV) or the whole tract (portal-drained viscera, PDV). Increasing the intake of alfalfa pellets from 800 to 1,200 g/d, while increasing the absorption and net flux across the MDV and PDV of free amino acids, had no effect on peptide absorption. Preparation of blood and plasma samples for peptide analysis with methods used in studies in which substantial peptide absorption has been reported indicated no net MDV or PDV flux of peptide. Such conflicting data on the extent of gastrointestinal peptide flux are discussed in the context of methodological differences and the importance of diet and physiological state of the animal.

Amino acid use by the gastrointestinal tract of sheep given lucerne forage.

Essential amino acid (EAA) utilization by gastrointestinal tract (GIT) tissues has been investigated in sheep given 800 and 1,200 g/day lucerne pellets. Animals prepared with indwelling catheters into the aorta and the portal drained viscera plus cannulas into the small intestine were infused with mixed U-13C-labeled amino acid or (1-13C]leucine tracers into the jugular vein or directly into the small intestine. GIT sequestration of EAA from arterial and luminal AA pools was determined from tracer and tracee arterioportal concentration differences at both levels of intake. Proportional tracer 13C-labeled EAA extraction of the arterial supply, on first pass across the GIT during jugular infusion, ranged from 0.063 for histidine to 0.126 for leucine. Recovery of intestinally infused tracer 13C-EAA at the portal vein ranged from 0.61 for histidine to 0.83 for valine. These data were independent of intake. Calculated rates of tracee sequestration by GIT tissues represented 0.45-0.65 of whole body EAA flux, except for histidine, for which the values were much lower (0.25-0.32). With the exception of phenylalanine, more than 0.8 of the EAA used by the GIT was extracted from circulating blood, thus calling into question the theory that GIT tissues make preferential use of EAA during absorptive metabolism, restricting supply to peripheral tissues such as skeletal muscle (growth) or mammary glands (lactation). Instead the GIT seems to compete very successfully with these tissues for circulating blood EAA.

Effect of intravenous amino acid infusion on leucine oxidation across the mammary gland of the lactating goat.

Changes in the kinetics of leucine in the mammary gland were examined in four lactating goats (25, 38, 45, and 135 DIM) that were given an i.v. infusion of a mixture of 18 AA, not including leucine, to alter the availability of leucine to the gland relative to other AA. Arteriovenous monitoring of [1-13C]leucine kinetics across one-half of the mammary gland was conducted on the last day (d 6 or 7) of the saline (control) and the AA infusion periods. Although blood flow to the mammary gland and the arterial concentration of most AA other than leucine were increased by the AA infusion, milk and protein yields did not change. For goats in early lactation (n = 3), arterial leucine concentrations fell considerably during AA infusion; however, the arteriovenous difference of leucine was maintained, resulting in uncommonly low leucine concentrations in venous plasma (8 microM). Whole body leucine flux (protein synthesis plus oxidation) was unaffected by AA infusion, but, because whole body leucine oxidation was reduced, whole body utilization of leucine for protein synthesis increased. The AA infusion reduced mammary oxidation of leucine to approximately one-third of control values. These results suggest that leucine oxidation can be reduced considerably without affecting milk protein output; thus, leucine oxidation may not be an irrevocable consequence of mammary metabolism. If catabolism of other AA either by the gland or in the whole body can be reduced, then the efficiency of milk yield can be improved.

Evidence for the utilization of peptides for milk protein synthesis in the lactating dairy goat in vivo.

Precursors for milk protein synthesis have been examined in lactating dairy goats using arteriovenous difference and isotope kinetic techniques. Certain amino acids, such as phenylalanine and histidine, are taken up by the mammary gland in quantities that are insufficient to account for their output in milk protein. Some amino acids have been shown to be present in significant quantities (10-30% of total non-protein-bound amino acids) as peptides (< 1,500 Da) in the arterial supply to the mammary gland, although methodological considerations make it difficult to accurately assess the extent of their uptake across the tissue bed. Indirect evidence for the utilization of peptides for milk protein synthesis in vivo has been obtained, however, by examination of the kinetics of milk casein labeling during long-term (24 h) systemic infusion of [1-13C]phenylalanine and [1-13C]leucine. Comparison of plateau enrichments for blood, plasma, and casein indicate that, although, for leucine, the plasma free pool seems to provide all the leucine for milk protein synthesis, sources other than the labeled plasma free amino acids contribute phenylalanine (10-20%) for casein biosynthesis. These findings raise questions relating to the type and source of amino acid precursors used by tissues for protein synthesis.

Responses in milk constituents to intravascular administration of two mixtures of amino acids to dairy cows.

Four Holstein-Friesian cows were used to investigate the effects of intravascular infusions of AA mixtures on milk constituents. Cows were in wk 11 to 28 of lactation and were fed a basal concentrate (142 g of CP/kg of DM) and grass silage (149 g of CP/kg of DM) in a 60:40 ratio (percentage of DM). Cows were fed hourly, and feed intake was fixed at 95% of ad libitum intake for each experimental period. Each cow received a 4-d jugular saline infusion, followed by a 5-d jugular infusion of a mixture of AA. Two mixtures of AA were used in a crossover design. The first mixture contained both the essential AA and non-essential AA found in milk protein (total AA); this mixture was infused at 400 g of AA/d. The other mixture represented the essential AA fraction only and was infused at 208 g/d. Infusion of total AA increased milk protein concentration from 32.4 to 35.0 g/kg, and essential AA increased milk protein concentration from 32.5 to 36.9 g/kg; milk protein yield increased by 87 g/d (total AA) and 143 g/d (essential AA). Intravascular administration of AA specifically stimulated milk protein concentration, and the efficiency with which the AA were used was higher than had been previously reported when AA supply was increased either by dietary supplementation or by abomasal infusion.

Leucine and protein metabolism in the lactating dairy cow mammary gland: responses to supplemental dietary crude protein intake.

Mammary gland protein metabolism, determined by an arteriovenous difference technique, was monitored in four Holstein-Friesian dairy cows in response to supplemental dietary protein (provided as rumen-protected soyabean meal) during late lactation (weeks 24-30). Each cow was offered two isoenergetic diets composed of grass silage (170 g crude protein/kg dry matter) plus either a low (108 g/kg) or medium (151 g/kg) crude protein concentrate in a single crossover design involving two 21 d periods. On day 21, arteriovenous measurements across the mammary gland were made during a 13 h continuous i.v. infusion of [1-(13)C]leucine and with frequent (2 hourly) milk sampling during the final 6 h. Although total milk yield was slightly increased (+1 kg/d) by protein supplementation, milk protein yield was not significantly affected. Whole body protein flux (protein synthesis plus oxidation) was not significantly affected by supplementation. Total mammary gland protein synthesis (milk plus non-milk protein) was also not affected by supplementation but on both diets gland synthesis was always greater (by 20-59 percent) than milk protein output. The fractional oxidation rate of leucine by the mammary gland was significantly increased by protein supplementation (0-047 v. 0-136). Although the enrichment of leucine in secreted milk protein continued to increase, the final value (at 13 h) was 0-94 of the arterial plasma free leucine plateau value (not significantly different), suggesting almost exclusive use of plasma free leucine for milk protein synthesis. Based on current feeding schemes for dairy cattle, a fixed proportion (0-65 0-75) of the additional protein intake (+490 g/d) should have been partitioned into milk protein. Instead, leucine oxidation by the mammary gland was increased. Whether oxidation of other amino acids was also enhanced is unknown but if amino acid oxidation and the 'additional' non-milk protein synthesis occurring in the gland are not crucial to milk synthesis, then by reducing such activities improvements in the efficiency of converting absorbed amino acid into milk protein can be achieved.

The effect of dietary crude protein as protected soybean meal on mammary metabolism in the lactating dairy cow.

Metabolism in the mammary gland was related to changes in milk output in response to changes in dietary protein intake. Three diets of grass silage and concentrate were fed to four lactating dairy cows equipped with intravascular catheters across the mammary gland. Concentrates differed in the inclusion of protected soybean meal and provided 11.3, 15.4, and 20.1% CP, respectively. Blood samples were taken to assess the effect of protein percentage on the nutrient fluxes across the gland and their relationship to milk production. Milk production, milk protein yield, and milk protein concentration were all increased as CP intake increased, although these responses were not linear. Concentrations of urea in milk reflected those in plasma and increased as dietary protein intake increased. Uptake of glucose and BHBA by the mammary gland tended to increase as milk production increased. Arterial supply of essential AA increased as the dietary protein increased. Supply and uptake of nonessential AA were unchanged by dietary treatment, and uptake was insufficient to account for output of nonessential AA residues in milk protein. The supply of essential AA was not limiting for milk protein synthesis, and some alternative mechanism must have existed for the control of milk protein yield.

Utilization of dipeptides by the caprine mammary gland for milk protein synthesis.

Specific use by the mammary gland in vivo of amino acids (AA) of peptide origin has been demonstrated in lactating dairy goats using a dual-labeled tracer technique involving close-arterial (external pudic artery, EPA) infusion of 13C-labeled dipeptides. The extent of utilization does not appear to differ for glycyl-L-[1-13C]phenylalanine and glycyl-L-[1-13C]leucine, perhaps indicative of a common mechanism by which AA are incorporated from peptide into milk protein. [1-13C]phenyl-alanine of peptide origin appears to be concentrated within the red blood cell, suggesting a role for the erythrocyte in peptide metabolism in vivo. In conclusion, it appears that the lactating mammary gland of goats has the ability to utilize AA of peptide origin for milk protein synthesis, and while the mechanism by which [1-13C]AA are incorporated into milk protein is not clear, it may involve peptide hydrolysis by either mammary cell surface or red blood cell hydrolases followed by uptake of liberated AA by the mammary gland.