Whole-body and splanchnic amino acid metabolism in sheep during an acute endotoxin challenge.

Supplemented protein or specific amino acids (AA) are proposed to help animals combat infection and inflammation. The current study investigates whole-body and splanchnic tissue metabolism in response to a lipopolysaccharide (LPS) challenge with or without a supplement of six AA (cysteine, glutamine, methionine, proline, serine and threonine). Eight sheep were surgically prepared with vascular catheters across the gut and liver. On two occasions, four sheep were infused through the jugular vein for 20 h with either saline or LPS from Escherichia coli (2 ng/kg body weight per min) in a random order, plus saline infused into the mesenteric vein; the other four sheep were treated with saline or LPS plus saline or six AA infused via the jugular vein into the mesenteric vein. Whole-body AA irreversible loss rate (ILR) and tissue protein metabolism were monitored by infusion of [ring-2H2]phenylalanine. LPS increased (P<0·001) ILR (+17 %), total plasma protein synthesis (+14 %) and lymphocyte protein synthesis (+386 %) but decreased albumin synthesis (-53 %, P=0·001), with no effect of AA infusion. Absorption of dietary AA was not reduced by LPS, except for glutamine. LPS increased the hepatic removal of leucine, lysine, glutamine and proline. Absolute hepatic extraction of supplemented AA increased, but, except for glutamine, this was less than the amount infused. This increased net appearance across the splanchnic bed restored arterial concentrations of five AA to, or above, values for the saline-infused period. Infusion of key AA does not appear to alter the acute period of endotoxaemic response, but it may have benefits for the chronic or recovery phases.

Responses in whole-body amino acid kinetics to an acute, sub-clinical endotoxin challenge in lambs.

Some effects of parasitism, endotoxaemia or sepsis can be mitigated by provision of extra protein. Supplemented protein may encompass a metabolic requirement for specific amino acids (AA). The current study investigates a method to identify and quantify the amounts of AA required during inflammation induced by an endotoxin challenge. One of each pair of six twin sheep was infused in the jugular vein for 20 h with either saline (control) or lipopolysaccharide (LPS, 2 ng/kg body weight per min) from Escherichia coli. Between 12 and 20 h a mixture of stable isotope-labelled AA was infused to measure irreversible loss rates. From 16 to 20 h all sheep were supplemented with a mixture of unlabelled AA infused intravenously. Blood samples were taken before the start of infusions, and then continuously over intervals between 14 and 20 h. At 20 h the sheep were euthanised, and liver and kidney samples were taken for measurement of serine-threonine dehydratase (SDH) activity. LPS infusion decreased plasma concentrations of most AA (P<0·05; P<0·10 for leucine and tryptophan), except for phenylalanine (which increased P=0·022) and tyrosine. On the basis of the incremental response to the supplemental AA, arginine, aspartate, cysteine, glutamate, lysine (tendency only), glycine, methionine, proline, serine and threonine were important in the metabolic response to the endotoxaemia. The AA infusion between 16 and 20 h restored the plasma concentrations in the LPS-treated sheep for the majority of AA, except for glutamine, isoleucine, methionine, serine and valine. LPS treatment increased (P<0·02) SDH activity in both liver and kidney. The approach allows quantification of key AA required during challenge situations.

The effect of medicated diets and level of feeding on caecal microbiota of lactating rabbit does.

AIMS: To study the effect of the type of antibiotic used in medicated diets against pathogens and the feeding level on the microbial biodiversity in the rabbit caecum. METHODS AND RESULTS: Three groups of eight does were given a diet unsupplemented (NAB) or with 100 ppm of bacitracin (BAC) or tiamulin (TIA). Litter sizes of four does in each group were adjusted to five (LS5) or to nine (LS9), to manipulate their levels of feed intake. The feeding level strongly affected caecal microbiota in does fed on NAB and BAC diet, whereas the effect of the antibiotic was higher in TIA-supplemented animals, even prevailing over the effect of feeding level. Daily food intake and milk yield (P<0.05) and caecum weight (P<0.10) were higher in feeding of LS9 does. The total volatile fatty acid concentration was lower with BAC (P<0.05). CONCLUSIONS: The feeding level strongly affects caecal biodiversity in lactating does. The extent of the antibiotic effect depends on its nature, being significant with TIA but not with BAC. SIGNIFICANCE AND IMPACT OF THE STUDY: Changes in the feeding level promote different profiles of caecal microbiota. Therapeutic doses of TIA may affect caecal microbiota, whereas BAC would not reduce diversity.

Effect of glutamine supplementation on splanchnic metabolism in lactating dairy cows.

The suggestion that glutamine (Gln) might become conditionally essential postpartum in dairy cows has been examined through increased postruminal supply of Gln. Net nutrient flux through the splanchnic tissues and mammary gland was measured in 7 multiparous Holstein cows receiving abomasal infusions of water or 300 g/d of Gln for 21 d in a crossover design. Milk yield increased significantly (by 3%) in response to Gln supplementation, but the 2.4% increase in milk protein yield was not statistically significant. Glutamine treatment had no effect on portal or hepatic venous blood flows. Net portal appearance of Gln and Glu was increased by Gln supplementation, accounting for 83% of the infused dose with, therefore, only limited amounts available to provide additional energy to fuel metabolism of the portal-drained viscera. The extra net portal appearance of Gln was offset, however, by a corresponding increase in hepatic removal such that net Gln splanchnic release was not different between treatments. Nonetheless, the Gln treatment resulted in a 43% increase in plasma Gln concentration. Infusions of Gln did not affect splanchnic flux of other nonessential amino acids or of essential amino acids. Glutamine supplementation increased plasma urea-N concentration and tended to increase net hepatic urea flux, with a numerical increase in liver hepatic O2 consumption. There were no effects on glucose in terms of plasma concentration, net portal appearance, net liver release, or postliver supply, suggesting that Gln supplementation had no sparing effect on glucose metabolism. Furthermore, mammary uptake of glucose and amino acids, including Gln, was not affected by Gln supplementation. In conclusion, this study did not support the hypothesis that supplemental Gln would reduce glucose utilization across the gut or increase liver gluconeogenesis or mammary glutamine uptake to increase milk protein synthesis.

Effect of postruminal glutamine supplementation on immune response and milk production in dairy cows.

Seventeen multiparous Holstein cows were used to examine the effect of an increased duodenal supply of Gln on immune function and production. Cows received continuous abomasal infusions of water (control: n = 8) or 300 g/d of Gln (n = 9) for 21 d starting within 48 h of calving. There were nonsignificant increases in milk and milk protein yields in response to Gln supplementation. Glutamine treatment had no effect on plasma glucose, nonesterified fatty acids (NEFA), or beta-hydroxybutyrate (BHBA) concentrations but did tend to increase plasma urea N concentration. The Gln treatment resulted in an increase of 108 microM in the plasma Gln concentration. Total essential AA concentrations decreased with the Gln treatment, whereas total nonessential AA concentrations were unaffected. T Lymphocyte proliferation did not differ between the control and Gln-treated cows. Treatment had no effect on the relative abundance of CD8 T cells but did increase the abundance of CD4 T cells. Cytokine production, as measured by IFN-gamma concentration determined in vitro in concanavalin-A-stimulated peripheral blood mononuclear cells, was similar between the treatments. Over the first 3 wk following calving, Gln supplementation had limited effects on milk production, metabolic parameters, and immune function.

Effects of dietary supplements of folic acid and rumen-protected methionine on lactational performance and folate metabolism of dairy cows.

The present experiment was undertaken to determine the interactions between dietary supplements of folic acid and rumen-protected methionine on lactational performance and on indicators of folate metabolism during one lactation. Fifty-four multiparous Holstein cows were assigned to 9 blocks of 6 cows each according to their previous milk production. Within each block, 3 cows were fed a diet calculated to supply methionine as 1.75% metabolizable protein, equivalent to 70% of methionine requirement, whereas the 3 other cows were fed the same diet supplemented with 18 g of a rumen-protected methionine supplement. Within each diet, the cows received 0, 3, or 6 mg/d of folic acid per kg of body weight. Rumen-protected methionine increased milk total solid concentration but not yield. Supplementary folic acid increased crude protein and casein concentrations in milk of cows fed no supplementary methionine and the effect increased as lactation progressed; it also decreased milk lactose concentration. Folic acid supplements had the opposite effects on milk crude protein, casein, and lactose concentrations in cows fed rumen-protected methionine. Milk and milk component yields and dry matter intake were unchanged. Folic acid supplementation increased serum folates and this response was greater at 8 wk of lactation. It decreased serum cysteine in cows fed rumen-protected methionine, whereas it had no effect in cows fed no supplementary methionine. The highest serum concentrations of cysteine but the lowest of vitamin B(12) were observed at 8 wk of lactation. Serum clearance of folic acid following an i.v. injection of folic acid was slower at 8 wk of lactation. During this period, the high concentrations of serum folates and cysteine, the low serum concentrations of vitamin B(12) and methionine, and the slow serum clearance of folates strongly suggest that the vitamin B(12) supply was inadequate and interfered with folate use. It could explain the limited lactational response to supplementary folic acid observed in the present experiment.

Glutamine in animal science and production.

With its many proposed metabolic roles, glutamine would seem to have major potential in normal animal production systems as well as during situations involving adverse challenges. In practice, however, responses to glutamine supplementation have been inconsistent. Thus, during lactation and growth studies in ruminants, both positive and null effects on production responses have been reported. Similarly, therapeutic responses to glutamine supplementation during various digestive tract disorders have been inconsistent in both pigs and ruminants. This is despite a proven involvement in the nucleic acid biosynthesis necessary to support cell proliferation. In sheep, at least, glutamine may exert a protective effect against hepatic amino acid (AA) oxidation, particularly for methionine. This may offer anabolic potential because methionine is the first limiting AA in a number of animal feedstuffs. Glutamine is also important in control of metabolic acidosis, but, in contrast to rodents, the main site of production seems to be extra-hepatic. In the immune system, while lymphocyte proliferation is glutamine-dependent, intracellular concentrations are low (in contrast to other tissues, such as muscle and liver). Instead, glutamate is accumulated, but the majority of this (approximately 65%) is derived in vivo from plasma glutamine. In sheep, endotoxin challenge elevates the plasma flux of glutamine, with a corresponding decrease in plasma concentration. At the same time, both the glutamate accumulation and fractional rate of protein synthesis within lymphocytes are enhanced. These lymphocyte responses, however, are not altered by an AA supplement that contains glutamine. Overall, although glutamine obviously plays important metabolic roles within the body, supplementation does not appear to provide consistent beneficial or therapeutic effects, except during certain catabolic situations. Glutamine availability, therefore, does not seem to be a limitation in many challenge situations. Rather, glutamine may signal alterations in nutrient demands among organs and a better understanding of this role may increase understanding of where modulation of glutamine status would be beneficial.

Effect of abomasal glucose infusion on alanine metabolism and urea production in sheep.

The effect of abomasal infusion of glucose (120 kJ/d per kg body weight (BW)0.75, 758 mmol/d) on urea production, plasma alanine-N flux rate and the conversion of alanine-N to urea was studied in sheep offered a low-N diet at limited energy intake (500 kJ/d per kg BW0.75), based on hay and grass pellets. Glucose provision reduced urinary N (P = 0.040) and urea (P = 0.009) elimination but this was offset by poorer N digestibility. Urea-N production was significantly reduced (822 v. 619 mmol/d, P = 0.024) by glucose while plasma alanine-N flux rate was elevated (295 v. 342 mmol/d, P = 0.011). The quantity of urea-N derived from alanine tended to be decreased by glucose (127 v. 95 mmol/d) but the fraction of urea production from alanine was unaltered (15%). Plasma urea and alanine concentrations (plus those of the branched chain amino acids) decreased in response to exogenous glucose, an effect probably related to enhanced anabolic usage of amino acids and lowered urea production.

Responses in the absorptive phase in muscle and liver protein synthesis rates of growing rats.

The effect of time after beginning of a meal (30, 60, 90 and 120 min) on liver and gastrocnemius muscle protein synthesis was tested in growing male rats using the large dose technique, based on a 10 min exposure to [15N]phenylalanine. The fractional synthesis rate was estimated from the ratio between the atom percent excess of tissue protein-bound and free labelled phenylalanine. The latter was measured by gas chromatography mass spectrometry using the tertiary-butyldimethylsilyl amino acid derivatives. The protein-bound phenylalanine of gastrocnemius muscle was separated from the other amino acids using preparative amino acid chromatography and then oxidised to N2 in an automated carbon-nitrogen Roboprep (CN) combustion module attached to a continuous flow isotope ratio mass spectrometer (IRMS), with m/z ions 28 and 29 monitored. The protein-bound phenylalanine from liver was separated by a gas chromatograph attached to a sample preparation module and an isotope ratio mass spectrometer (GC C-IRMS), with again m/z ions of 28 and 29 monitored. The following results were obtained: the daily fractional protein synthesis rates (ks) in gastrocnemius muscle and liver were 13.9% and 65.6% respectively, in 12 h fasted 145 g rats. These ks increased within 30 min after ingestion of meal to 14.9% and 91.8% for muscle and liver, respectively, and remained at these values for the next 90 min (14.6% and 87.4% at 60 min, and 14.3% and 88.6% at 120 min after the beginning of feeding). It was concluded that measurement of protein synthesis rates characteristics for the absorptive phase can be undertaken in a period from thirty minutes to two hours after start of a meal, without significant changes in the ks values.

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 importance of transmethylation reactions to methionine metabolism in sheep: effects of supplementation with creatine and choline.

The influence of administering the methylated products choline and creatine on methionine irreversible-loss rate (ILR) and recycling from homocysteine has been investigated in sheep fed close to energy and N equilibrium. Two methods to estimate methionine recycling were compared. The first involved [U-13C]methionine infused as part of a labelled amino acid mixture obtained from hydrolysed algal protein. In this approach the isotope dilution of methionine with all five C atoms labelled (m + 5) will represent the ILR which does not recycle through homocysteine, while that which includes molecules with C-1-C-4 labelled will allow for loss of the labelled methyl (5)-C atom and replacement by an unlabelled moiety in the remethylation of homocysteine. The second method involved a combined infusion of [1-13C]- and [S-methyl-2H3]methionine. These two approaches gave similar data for methionine ILR which does not include label recycled to the amino acid from homocysteine but differed for recycled methionine fluxes. Consequently the two procedures differed in the calculated extent of homocysteine methylation under control conditions (6 v. 28%). These extents of remethylation are within the range observed for the fed human subject, despite the fact that fewer dietary methyl groups are available for the ruminant. Using combined data from the infusions, significant depression of methionine recycling occurred in blood (P < 0.05), with a similar trend for plasma (P = 0.077), when choline plus creatine were infused. Wool growth, assessed by intradermal injection of [35S]cysteine, was not altered by supplementation with the methylated products. From changes in the label pattern of free methionine in aortal, hepatic portal and hepatic venous blood during U-13C-labelled algal hydrolysate infusion, the major sites of homocysteine remethylation appear to be the portal-drained viscera and the liver. This was confirmed by analysis of free methionine enrichments in various tissues following dual infusion of [1-13C]- and [S-methyl-2H3]methionine, with the greatest activities occurring in rumen, jejunum and liver. Of the non-splanchnic tissues examined, only kidney exhibited substantial methionine cycling; none was detected in muscle, heart, lung and skin. The implications of methyl group provision under net production conditions are discussed.

Protein synthesis in splanchnic tissues of sheep offered two levels of intake.

Protein synthesis rates were measured in liver and gastrointestinal tract (GIT) sections of fattening sheep offered lucerne (Medicago sativa) pellets at either 1.25 or 2 times energy maintenance. The measurement technique involved a large dose of [1-13C]valine over 60 min. Animals on the higher intake had a larger mass of liver protein (143 v. 100 g, P = 0.02), similar fractional synthesis rates (ks; 22.5 v. 22.1%/d, not significant) and greater absolute amounts of protein synthesis (32 v. 23 g/d; P = 0.016) compared with those on the smaller amount of ration. The ks values and RNA: protein in the GIT sections also tended to increase with food intake. Estimated total GIT protein synthesis was approximately three-fold that in liver and probably constituted 25-35% of whole body synthesis. All splanchnic tissues measured had lower translational efficiencies (g protein synthesized/d per g total RNA) than reported for milk-fed and newly-weaned lambs and this may relate to the decline in the rate of protein deposition as lambs progress to the fattening condition.

Protein synthesis in ovine muscle and skin: sequential measurements with three different amino acids based on the large-dose procedure.

1. Protein synthesis has been determined in biopsies from ovine skin and muscle by sequential use of three [13C] amino acids, valine leucine and phenylalanine, as large-dose injections. 2. Leucine and phenylalanine increased plasma insulin concentrations within 40 min of injection. 3. All three amino acids decreased the plasma concentrations of other amino acids. 4. Intracellular free amino acids in muscle decreased while those in skin increased. 5. The fractional rates of protein synthesis were similar, regardless of which amino acid was used, although the rates for muscle were significantly less than for skin (2.1 vs 11.0%/day, P < 0.001).

Responses in tissue protein synthesis to sub- and supra-maintenance intake in young growing sheep: comparison of large-dose and continuous-infusion techniques.

In ten lambs (average live weight 33 kg), five offered 300 g/d (approximately 0.6 x maintenance; L) and five 900 g/d (1.8 x maintenance; H), tissue protein synthesis was measured by three procedures simultaneously. The techniques involved continuous infusion of [U-14C]phenylalanine and [1-13C]leucine over 7-8 h followed by a terminal large dose of [15N]phenylalanine during the last 30 or 60 min. Rates of protein synthesis were then calculated based on the free amino acid or oxo-acid isotopic activity in either arterial, iliac venous blood or tissue homogenate for the continuous-infusion studies, or on plasma or tissue homogenate for the large-dose procedure. For muscle (> 99%), and to a lesser extent skin (85-93%), effective flood conditions were achieved with the [15N]phenylalanine but were either not established or maintained for liver and tissues of the gastrointestinal tract (< 50%). The large dose of phenylalanine also caused changes in the concentration and isotopic activity of blood leucine and 4-methyl-2-oxo-pentanoate. Based on the assumption that the large-dose procedure yields the closest value for the true rate of protein synthesis (L 1.97%/d, H 2.85%/d) then, for muscle, only values based on the homogenate as precursor gave comparable results for both leucine (L 1.83%/d, H 3.01%/d) and phenylalanine (L 1.67%/d, H 2.71%/d) continuous infusion. The values based on the arterial or venous amino or oxo-acid were significantly less, more so at the lower intake. In contrast, for skin, a tissue dominated by export protein synthesis, values from the large-dose procedure (L 6.37%/d, H 10.98%/d) were similar to those derived with arterial or venous metabolites as precursor (L 5.23 and 6.93%/d, H 9.98 and 11.71%/d for leucine), but much less than those based on homogenate data. Based on the large-dose technique, protein synthesis increased with intake in muscle (P < 0.001), skin (P = 0.009) and liver (26.7 v. 30.5%/d; P = 0.029). The contributions of muscle and skin to total protein synthesis were approximately equal. The incremental efficiency of conversion for muscle of synthesized protein into deposition appeared to be similar to values reported for rodents.