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Changes in splanchnic energy and N metabolism were studied in sheep, prepared with vascular catheters across the portal-drained viscera (PDV) and the Liver, and maintained on supramaintenance intakes of either grass or grass + barley pellets. The animals were challenged, on both diets, with 4 d intra- mesenteric vein infusions of NH4CI (25 µmol/min) plus NH4HCO3(at either 0 or 125 µmol/min). On the final day of each treatment the natural abundance NH4Cl was replaced with15NH4Cl over a 10 h infusion while over the same period [l-13C]leucine was infused via a jugular vein. Measurements were made of blood flow plus mass transfers of NH3, urea, free amino acids and O2, across the PDV and liver. Enrichments of [14N15N]urea and [15N15N]urea plus [15N]glutamine, aspartate and glutamate were also monitored. Whole-body urea flux was determined by infusion of [14C]urea. At the end of the study the animals were infused for 3 h with15NH4CI, killed and liver samples assayed for intracellular free amino acid enrichments and concentrations. Blood flows across the splanchnic region were unaffected by either diet or level of ammonium salt infusion. At the lower ammonium salt infusion there was a trend for greater absorption of NH3across the PDV (P3. The PDV contributed 19–28% to whole-body O2consumption and the liver 23–32%. Hepatic extraction of absorbed NH3was complete on all treatments and systemic pH remained constant. The fractions of urea-N apparently derived from NH3, were similar on the grass (0·59–0·64) and grass + barley (0·64–0·67) diets. Hepatic production of urea agreed well with urea flux measurements. Between the two levels of ammonium salt infusion and within diets the additional NH3removed across the PDV was accounted for by the increased urea-N production. The [14N15N]: [15N15N] ratio of the urea produced was 97:3, while the enrichment of hepatic intracellular free aspartate was lower than that of [14N15N]urea. Glutamine enrichments were 0·23–0·37 those of [14N15N]urea, indicating a minor role for those hepatocytes (probably perivenous) which contain glutamine synthetase (EC6.3.1.2). Leucine kinetics, either for the whole body or splanchnic tissues, were not different between diets or level of ammonium salt infusion, except for oxidation which was less on the grassfbarley ration. Amino acid concentrations were lower on the grass + barley diet but net PDV absorptions were similar. The pattern of essential amino acids absorbed into the PDV showed good agreement with the published composition of mixed rumen microbial protein. Fractional disappearances of absorbed free essential amino acids across the liver varied from 0·4 (branched chains) to near unity (histidine, phenylalanine)

The response in whole-body and splanchnic tissue mass and isotope amino acid transfers in both plasma and blood has been studied in sheep offered 800 g lucerne (Medicago sutiva) pellets/d. Amino acid mass transfers were quantified over a 4 h period,by arterio-venous procedures, across the portal-drained viscera (PDV) and liver on day 5 of an intravenous infusion of either vehicle or the methylated products, choline (0.5 g/d) plus creatine (10 g/d). Isotopic movements were monitored over the same period during a 10 h infusion of a mixture of U-13C-labelled amino acids obtained from hydrolysis of labelled algal cells. Sixteen amino acids were monitored by gas chromatography-mass spectrometry, with thirteen of these analysed within a single chromatographic analysis. Except for methionine, which is discussed in a previous paper, no significant effects of choline plus creatine infusion were observed on any of the variables reported. Whole-body protein irreversible-loss rates ranged from 158 to 245 g/d for the essential amino acids, based on the relative enrichments (dilution of the U-13C molecules by those unlabelled) of free amino acids in arterial plasma, and 206-519 g/d, when blood free amino acid relative enrichments were used for the calculations. Closer agreement was obtained between lysine, threonine, phenylalanine and the branched-chain amino acids. Plasma relative enrichments always exceeded those in blood (P < 0.001), possibly due to hydrolysis of peptides or degradation of protein within the erythrocyte or slow equilibration between plasma and the erythrocyte. Net absorbed amino acids across the PDV were carried predominantly in the plasma. Little evidence was obtained of any major and general involvement of the erythrocytes in the transport of free amino acids from the liver. Net isotope movements also supported these findings. Estimates of protein synthesis rates across the PDV tissues from [U-13C] leucine kinetics showed good agreement with previous values obtained with single-labelled leucine. Variable rates were obtained between the essential amino acids, probably due to different intracellular dilutions. Isotope dilution across the liver was small and could be attributed predominantly to uni-directional transfer from extracellular sources into the hepatocytes and this probably dominates the turnover of the intracellular hepatic amino acid pools.

The effects of either low (25 μmol/min) or high (235 μmol/min) infusion of NH4Cl into the mesenteric vein for 5 d were determined on O2consumption plus urea and amino acid transfers across the portal-drained viscera (PDV) and liver of young sheep. Kinetic transfers were followed by use of15NH4Cl for 10 h on the fifth day with simultaneous infusion of [1-13C]lleucine to monitor amino acid oxidation. Neither PDV nor liver blood flow were affected by the additional NH3loading, although at the higher rate there was a trend for increased liver O2consumption. NH3-N extraction by the liver accounted for 64–70% of urea-N synthesis and at the lower infusion rate the additional N required could be more than accounted for by hepatic removal of free amino acids. At the higher rate of NH3administration additional sources of N were apparently required to account fully for urea synthesis. Protein synthesis rates in the PDV and liver were unaffected by NH3infusion but both whole-body (P< 0·05) and splanchnic tissue leucine oxidation were elevated at the higher rate of administration. Substantial synthesis of [15N]glutamine occurred across the liver, particularly with the greater NH3supply, and enrichments exceeded considerably those of glutamate. The [15N]urea synthesized was predominantly as the single labelled, i.e. [14N15N], species. These various kinetic data are compatible with the action of ovine hepatic glutamate dehydrogenase (EC1.4.1.2) in periportal hepatocytes in the direction favouring glutamate deamination. Glutamate synthesis and uptake is probably confined to the perivenous cells which do not synthesize urea. The implications of NH3detoxification to the energy and N metabolism of the ruminant are discussed.

1. 1. Protein synthesis has been determined in biopsies from ovine skin and muscle by sequential use of three [ 13 C] amino acids, valine leucine and phenylalanine, as large-dose injections. 2. 2. Leucine and phenylalanine increased plasma insulin concentrations within 40 min of injection. 3. 3. All three amino acids decreased the plasma concentrations of other amino acids. 4. 4. Intracellular free amino acids in muscle decreased while those in skin increased. 5. 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

Forty-six Simmental × British Friesian bull calves were allocated to six treatment groups. In four groups (each of eight animals) half the animals were given a prime injection of gonadotropin-hormone-releasing-hormone (GnRH) either as the decapeptide or as an octapeptide (residues 3 to 10) conjugated to egg albumen. Prime injection times were at 3, 4, 5 or 6 months of age. All animals were boosted with a GnRH conjugate, similar to that used for the prime injection, at 8 months. At 8 months, six other animals were surgically castrated while the remaining eight were left as untreated bull controls. Weight gain and consumption of a barley-based diet offered ad libitum were recorded for individual animals. Blood samples were taken at a minimum of fortnightly intervals and the serum analysed for antibody titre against GnRH, testosterone and insulin-like growth factor 1 (IGF-1). Animals were slaughtered at 12 months and chemical analyses performed on the dissectible material of the 10th rib for protein, lipid, ash and water content. Greater antibody titres and a longer period of low serum testosterone were achieved with the octapeptide conjugate compared with the decapeptide. Serum IGF-1 slowly decreased following both surgical- and effective immuno-castration. There were no significant differences in food intake between the groups. Both steers and the more responsive immunocastrates had higher fat (P < 0·01), lower protein (P < 0·05) and water concentrations (P < 0·01) in tissues from a rib sample joint compared with untreated bulls. As immuno-responsiveness decreased there were indications of compensatory changes in body composition. The technique may be applicable during periods of, for example, mixed grazing for bulls.

Whole-body protein synthesis, estimated by the irreversible loss rate procedure, and hind-leg protein metabolism determined by arterio-venous techniques were monitored in response to three nutritional conditions (approximately 0.6, 12 and 1.8 x energy maintenance (M)) in ten wether lambs (33 kg average live weight). In all lambs and treatments measurements were based on radiolabelled phenylalanine, but the terminal procedures (five at 0.6 x M and five at 1.8 x M) also included infusion of [1-13C]leucine; this permitted comparison of amino acids catabolized (leucine) and non-metabolized (phenylalanine) by the hind-limb tissues. Whole-body protein synthesis increased with intake and the relationship with energy expenditure was slightly lower than that reported previously for pigs and cattle. The efficiency of protein retention: protein synthesis did not exceed 0.25 between the two intake extremes. Effects of intake on amino acid oxidation were similar to those observed for cattle. Hind-limb protein synthesis also increased significantly (P < 0.001) in response to intake. Estimates of protein gain, from net uptake values, indicated that the tissues made a greater proportional contribution to total protein retention above M and to protein loss below M, emphasizing the role played by muscle tissue in providing mobile protein stores. The rates of protein synthesis calculated depended on the selection of precursor (blood) metabolite, but rates based on leucine always exceeded those based on phenylalanine when precursor from the same pool was selected. The incremental efficiency of protein retained: protein synthesis was apparently unity between 0.6 and 1.2 x M but 0.3 from 1.2 to 1.8 x M. Blood flow through the iliac artery was also proportional to intake. Leucine and oxo-acid catabolism to carbon dioxide increased with intake such that the metabolic fate of the amino acid was distributed in the proportion 2:1 between protein gain and oxidation. The rates of oxidation were only 1–3% the reported capacity of the rate-limiting dehydrogenase enzyme in muscle, but sufficient enzyme activity resides in the hind-limb adipose tissue to account for such catabolism

A method has been developed based on stable isotopes and biopsy procedures which allows the large-dose procedure for measurement of protein synthesis to be applied in serial studies to farm species. Measurements of total nitrogen retention and protein synthesis in m. longissimus dorsi and m. vastus lateralis were made in five wether lambs (40–44 kg) infused intravenously, successively, with vehicle (10 d); testosterone (15 d; 9 mg/d); vehicle (15 d). N retention was improved by testosterone infusion (+2.9 g N/d; a 96% improvement total over control periods). Muscle protein synthesis was not significantly altered by exogenous hormone administration, nor were RNA:protein, RNA:DNA or protein: DNA. The implication of the developed procedure for dynamic studies in accessible tissues of large animals is discussed.

Rates of protein synthesis for the liver, plasma albumin and total plasma protein were quantified in sheep either offered a supra-maintenance intake or fasted for 3 d. The technique of continuous infusion over a 12 h period was employed with the simultaneous infusion of [1-13C]glycine, [1-13C]leucine and [2H5]phenylalanine. Blood and plasma samples were removed at timed intervals from the hepatic portal and hepatic veins plus the aorta. Enrichments of the free amino acids (AA) were determined in all blood and plasma samples as was the protein-bound AA in an apolipoprotein B100 extract. Protein-bound phenylalanine enrichments were also measured in albumin and total protein from plasma plus samples from liver biopsies. The apolipoprotein B100 enrichments agreed well with those of the free AA in hepatic (and hepatic portal) plasma but were lower than for arterial free AA and greater than liver homogenate free AA. This adds support to the concept that export proteins may preferentially use AA directly from extracellular sources. Intake had no significant effect on constitutive liver protein synthesis and the values agreed well with those obtained by other isotopic approaches. There were, however, signicant declines, based on hepatic venous free phenylalanine enrichment, at the lower intake in both the fractional (3·4v. 4·7 % per d;P=0·024) and absolute (2·4v. 4·2 g/d;P=0·011) synthesis rates of albumin, which matched the estimated decrease in total plasma albumin content (52v. 67 g,Pv. 151 g,P=0·035) with no observed significant difference in kinetic parameters. Albumin synthesis was calculated to account for a maximum of 17 % of total liver protein synthesis in the fed condition and this may fall to 8 % during moderate fasts.

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-l–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 [l-13C]- & [S-methyl-2H3& rsqb;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 infwions, significant depression of methionine recycling occurred in blood (P 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 & [1J3C& ]- and & [S-methyl-2H3methionine, 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 prduction conditions are discussed.

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 (26.7v.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

1. Five Greyface wethers (42–45 kg) fed on various fixed amounts of dried grass pellets (either approximately 1·3 times maintenance or 2 times maintenance) by means of belt-type continuous feeders were housed in opencircuit respiration chambers for periods of 45 d. Between days 15 and 35 they received daily oral doses of 1·5 mg of the β-adrenergic agonist clenbuterol (adsorbed on to the feed). Continuous energy and nitrogen balance measurements each of 5 d duration were conducted throughout the chamber confinement.2. On six occasions (twice during the 15 d pre-clenbuterol period, on days +4, +11 and +18 of clenbuterol administration and once during the post-treatment period) animals were infused with [l-14C]leucine to determine the rates of leucine oxidation and the amounts of leucine available for protein synthesis.3. Clenbuterol administration caused a marked increase in N retention (2–3 g N/d; P < 0·001) throughout the 20 d treatment period. It also increased (P < 0·001) the energy expenditure of the animals (on average by 1·1 MJ/d over the first 5 d, compared with immediate pretreatment values, and 0·6 MJ/d over the 20 d period, compared with the mean of pre- and post-treatment control values). The effect of treatment was calculated to result, on average, in the daily retention of 19 (SE 1·5) g more protein and 30 (SE 5·5) g less fat.4. During clenbuterol treatment leucine oxidation was reduced (P < 0·01). However, values for the amounts of leucine available for protein synthesis were equivocal, with an increase (P < 0·001) on day 11 of treatment, but no change on days 4 and 18.5. Withdrawal of the clenbuterol resulted in rapid alterations of N and energy metabolism towards those expected of control animals of that weight.

1. Eighteen New Zealand White × Califomian young female rabbits (does) were paired by weight and age and, at 800 g live weight, placed on a restricted intake based on the mean body weight of each pair. On attaining 1 kg one of each pair was then given 10 mg trenbolone acetate by injection on days 1, 3 and 5 of each week. This continued until the animals reached 2·3 to 2·4 kg (5 to 6 weeks).2. There were no differences in digestible or metabolizable energy intakes between treated and untreated rabbits. However, the treated animals showed small but significant increases in rates of gain for live and empty body weight compared with control does. Treated animals also had significantly more nitrogen (P < 0·001) and less energy (P < 0·05) per unit empty body weight than controls. Although the proportion of total nitrogen retention was 0·19 greater (P < 0·001) in treated does, total energy retention was only slightly less. Treated animals had approximately 40 g (200 g/kg) less fat and 50 g more protein (N × 6·25) than control does at the end of the trial.3. Increases in liver weight and clitoral size were observed in treated animals.

1. The effects of progressive reduction in food intake from 1.6 x maintenance (1.6 M) to approximately maintenance (M′) and then to zero (fasting) on energy expenditure and leucine kinetics were examined in Hereford x Friesian finishing beef steers.2. Estimates of whole body protein synthesis and protein oxidation were obtained from the specific radioactivity of free leucine in blood and of exhaled carbon dioxide during continuous infusions of [I-14C]leucine. Protein synthesis contributed a minimum of 0.19 to total heat production across all three intakes.3. The apparent efficiencies with which synthesized protein was retained were 0.28 between 1.6 M and M′ and 1.04 between M′ and fasting. The greater efficiency below M′ reflected probable use of amino acids as energy sources during fasting, which would be spared as soon as feed was available.4. Nitrogen derived from protein oxidation made a minor contribution to urine N at both 1.6 M (0.45) and M′ (0.36) but provided a significant proportion to the increment in urine N between intakes (0.68).5. Amino acid absorption, estimated indirectly as the sum of protein oxidation and protein retention, represented only 0.28 of N intake at M′ and 0.38 at 1.6 M but the contribution to the increment in N intake between these two diet levels was greater (0.56).6. The estimated efficiency of utilization of absorbed amino acids between M′ and 1.6 M was 0.45.

1. The effects on growth performance, energy and nitrogen retention, and leucine metabolism of a subcutaneous combined implant of 140 mg trenbolone acetate (TBA)+20 mg oestradiol-17β (OE) have been examined in Hereford × Friesian beef steers (280–520 kg). Comparisons were made both with the same animals before implantation and with untreated control animals maintained under similar physiological and nutritional conditions.2. Over a 10 week period the implanted steers showed an improvement in rate of live-weight gain (LWG) of 0.5–0.6 with an even greater proportional increase in N retention compared with control animals. Total energy retention was unaffected and thus the ratio, protein energy: total energy gain was 0.43 for implanted steers compared with 0.26 for untreated animals.3. Estimates of protein synthesis and protein oxidation were obtained from the specific radioactivities of blood free-leucine and exhaled carbon dioxide during continuous infusions of [1-14C]leucine. Whole-body protein synthesis, based on metabolic size, and amino acid fractional oxidation remained similar for control steers throughout the experiment. Steroid-treated steers showed a slight decline in synthesis which was significant (P < 0.05) at week + 5 post-implant while amino acid oxidation was significantly lower at weeks +2 (P < 0.01) and + 5 (P < 0.05) compared with control animals. The ratio, protein deposition: protein synthesis was 0.05 for control animals but 0.08–0.10 for steroid-treated animals after implantation.4. There was a slight decrease in urinary NT-methylhistidine elimination after implantation which suggested that muscle protein degradation may be reduced although the estimated decrease was insufficient to account for the total improvement in growth rate and N retention.5. The results suggest that for both control and treated steers, less than 0.5 of total urine N elimination was derived directly from tissue catabolism of protein and amino acids.6. The combined action of the exogenous steroids in the promotion of protein gain, primarily through a decrease in total protein degradation with little alteration of total energy retention, is compared with present understanding of the role of the endogenous sex hormones.

The effects of episodic infusion of testosterone into the vascular system on energy expenditure, nitrogen retention and whole body protein synthesis (determined from [1-14C]leucine kinetics) were studied in castrated male lambs under conditions of controlled food intake. Comparisons were made between a 10-day control period and a 10-day treatment period for each lamb. Infusion of testosterone produced a significant increase in heat production, but the magnitude (198 kJ/day, +2·5% was less than the differences in energy expenditure expected between entire and castrated male ruminants. The retention of nitrogen improved by 1·24 g/day ( + 22%) in response to the administration of androgen, and this was accompanied by a decrease in amino acid oxidation. Total protein synthesis also declined, and the anabolic nature of testosterone supply must, therefore, be effected through a reduction in the breakdown of protein, the mechanism being similar to that proposed for certain anabolic steroids and the β-agonist, clenbuterol. Contrary to other reports, the presence of testosterone had no effect on the plasma concentration of GH. J. Endocr. (1987) 115, 439–445

The specific radioactivity of 14 CO 2 (> 3 nCi mol −1 ) can be determined by conversion to an insoluble carbonate followed by re-evolution of the gas, using a mineral acid, through a proprietary organic sample oxidiser (Packard 306 Oxidiser, Packard Instruments Ltd, Caversham, Berks, U.K.).