Phytol supplementation alters plasma concentrations of formate, amino acids, and lipid metabolites in sheep.

The phytol moiety in chlorophyll molecules acts as an agonist of peroxisome proliferator-activated receptor-α in monogastric animals. The current study aimed to clarify the effects of dietary supplementation with phytol on the plasma concentrations of formate and amino acids related to one-carbon (1C) donors and its effects on lipid metabolism in sheep. Four mature sheep were fed with a mixed ration (metabolizable energy, 10.7 MJ/kg DM; CP, 150 g/kg DM) comprising barley, rice bran, soybean meal, and oat hay at 1.5 times maintenance metabolizable energy for three consecutive 14-day experimental periods. The first and third periods served as controls without phytol supplementation, while in the second period, phytol was added to the mixed ration at 12 g/kg of dietary DM per day. In each period, feces, urine, and jugular blood samples were collected. Dry matter intake in relation to metabolic BW was slightly lower (P < 0.01) in the first period than the second and third periods but did not differ between the latter two periods. Dry matter digestibility was slightly reduced (P = 0.05) by the phytol treatment. Nitrogen (N) intake and retention showed similar trends to DM intake, but urinary N was unchanged among the periods. Plasma cholesterol and phospholipid concentrations decreased during the phytol treatment period, while triglyceride concentration increased (P < 0.05). In the phytol treatment period, the plasma concentrations of serine and glycine (1C donors) increased, but the glutamate level decreased (P < 0.01). Plasma concentrations of formate and methionine increased (P < 0.01) from the first control period to the phytol supplementation period, but homocysteine and cysteine (intermediate and by-product of the methionine cycle) levels were unchanged among the treatment periods. In conclusion, dietary phytol affects lipid metabolism as well as amino acid metabolism and 1C donors in sheep. These effects may be associated with the activity of phytol as an agonist of the nuclear receptors, although this needs further investigation.

Response of plasma glucagon-like peptide-2 to feeding pattern and intraruminal administration of volatile fatty acids in sheep.

Glucagon-like peptide-2 (GLP-2), a gut peptide secreted by enteroendocrine L cells, has recently been identified as a key regulator of intestinal growth and absorptive function in ruminants. However, reports on GLP-2 secretion are few, and more information regarding its secretion dynamics is needed. In this study, two experiments were conducted to elucidate the daily rhythm of GLP-2 secretion in response to feeding regimen and to investigate the effect of volatile fatty acids (VFA) on GLP-2 release in sheep. In experiment 1, blood samples were collected over 3 d from 4 Suffolk mature wethers adapted to a maintenance diet fed once daily; day 1 sampling was preceded by 24 h of fasting to reach steady state. On days 1 and 3, samples were collected every 10 min from 11:00 to 14:00 on both days and then every 1 h until 00:00 on day 1 only; feed was offered at 12:00. On day 2, feed was withheld, and sampling was performed every hour from 01:00 to 00:00. In experiment 2, 5 Suffolk mature wethers were assigned to 5 treatment groups of intraruminal administration of saline, acetate, propionate, butyrate, or VFA mix (acetate, propionate, and butyrate in a ratio of 65:20:15) in a 5 × 5 Latin square design. Blood samples were collected at 0, 1.5, 3, 6, 9, 12, 15, 20, 25, 30, 40, 50, 60, 90, and 120 min relative to the beginning of administration at 12:00. In both experiments, plasma GLP-2, glucagon-like peptide-1 (GLP-1), glucose, insulin, and ß-hydroxy butyric acid (BHBA) levels were measured. In experiment 1, incremental area under the curve was greater (P < 0.05) post-feeding than pre-feeding on days 1 and 3 for GLP-2 and tended to be greater (P < 0.1) on day 1 for GLP-1. Plasma insulin, glucose, and BHBA levels increased (P < 0.05) on day 1 post-feeding. Plasma GLP-2 was poorly correlated with GLP-1 but positively correlated with insulin, glucose, and BHBA. In experiment 2, administration of butyrate and VFA mix remarkably increased plasma GLP-2 (P = 0.05) and BHBA (P < 0.0001) levels compared with those in other treatments. Plasma GLP-1 levels were higher with butyrate administration compared with those in the saline, acetate, and VFA mix (P = 0.019). Propionate administration increased plasma glucose (P = 0.013) and insulin (P = 0.053) levels. Thus, our data confirmed that GLP-2 release is responsive to feeding and might be promoted by BHBA produced by the rumen epithelial metabolism of butyrate. Further molecular- and cellular-level studies are needed to determine the role of butyrate as a signaling molecule for GLP-2 release.

Expression of lipases and lipid receptors in sperm storage tubules and possible role of fatty acids in sperm survival in the hen oviduct.

The aim of this study was to determine the role of fatty acids for sperm survival in the sperm storage tubules (SSTs) of the hen oviduct. The mucosa tissues of uterovaginal junction (UVJ) of White Leghorn laying hens with or without artificial insemination using semen from Barred Plymouth Rock roosters were collected. The lipid density in the epithelium of UVJ and SST was analyzed by Sudan black B staining. The expressions of genes encoding lipid receptors and lipases were assayed by polymerase chain reaction in UVJ mucosa and SST cells isolated by laser microdissection. Fatty acid composition was analyzed by gas chromatography, and sperm were cultured with or without the identified predominant fatty acids for 24 hours to examine their effect on sperm viability. The lipid droplets were localized in the epithelium of UVJ mucosa and SSTs. The expression of genes encoding very low-density lipoprotein receptor(VLDLR), low-density lipoprotein receptor (LDLR), and fatty acid translocase (FAT/CD36) were found in SST cells. Expression of genes encoding endothelial lipase (EL), lipase H (LIPH), adipose triglyceride lipase (ATGL), and lipoprotein lipase (LPL) were found in UVJ. In contrast, only ATGL was found in SST cells, and its expression was significantly upregulated after artificial insemination. In UVJ mucosal tissues, five fatty acids, namely myristic acid (C14), palmitic acid (C16), stearic acid (C18), oleic acid (C18:1n9), and linoleic acid (C18:2n6), were identified as predominant fatty acids. The viability of sperm cultured with 1 mM oleic acid or linoleic acid was significantly higher than the sperm in the control culture without fatty acids. These results suggest that lipids in the SST cells may be degraded by ATGL, and fatty acids including oleic acid and linoleic acid may be released into the SST lumen to support sperm survival.

Effects of fat-enriched diet and methionine on insulin sensitivity in lactating cows.

The hyperinsulinemic-euglycemic clamp (EGC) technique was used to investigate the effects of calcium salts of long-chain fatty acids (LCFA-Ca) and rumen-protected Met (RPM) on insulin sensitivity in the peripheral tissues of lactating cows. Six multiparous Holstein cows were used in a 3 × 3 Latin square experiment in each 14-d period. Dietary treatments were 0 (RPM0), 20 (RPM20), and 60 (RPM60) g/d of RPM, supplemented with a diet containing 1.5% of LCFA-Ca equal to 110% of the cows' ME requirement. And as a control for the 3 LCFA-Ca-containing diets, a dietary treatment without LCFA-Ca (Con) was also included. After a 10-d adaptation period, milk samples were collected for 4 d, and EGC experiments were performed on d 14 of each treatment period. Insulin solution was infused through a jugular vein catheter at a rate of 0.1, 0.2, 0.3, and 0.4 milliunits·kg BW-1·min-1 for 30 min and then at a rate of 0.5 milliunits·kg BW-1·min-1 for 60 min. Glucose solution was variably infused to maintain plasma glucose at steady state through the same catheter. Blood samples for measurements were taken using the contralateral catheter. Plasma total cholesterol, cholesterol ester, free cholesterol, and phospholipid concentrations in RPM0 and RPM20 were higher than those in Con, whereas the concentrations in RPM60 were low at the same degree of those in RPM0 (P < 0.05). Plasma Met concentration was greatest in RPM60 (P < 0.05). In the EGC experiment, the glucose infusion rate was greater in RPM60 than in RPM0 and RPM20 and an effective concentration of insulin resulting in 50% maximal glucose infusion rate was lower in RPM60 compared with RPM0 (P < 0.05), indicating that insulin sensitivity was intensified in RPM60. Although the insulin sensitivity evaluated from the EGC data in RPM0, RPM20, and RPM60 was not different from Con, a slight decline was observed in RPM0 and insulin sensitivity in RPM60 was higher than Con. Our results from the EGC experiment demonstrated that the feeding RPM lead to increased insulin sensitivity, which suggests that dietary Met affects lipid metabolism via insulin action in lactating dairy cows fed a LCFA-Ca-containing diet.

Ingestion of medium chain fatty acids by lactating dairy cows increases concentrations of plasma ghrelin.

The purpose of this study was to elucidate the effects of medium-chain fatty acids (MCFAs) on plasma ghrelin concentration in lactating dairy cows. Five early-lactating Holstein cows were randomly assigned to 2 dietary treatments in a crossover design with 2-wk periods. Treatments consisted of diets supplemented or not (control) with calcium salts of MCFAs (MCFA-Ca; 1.5% dry matter). Plasma hormone and metabolite concentrations in blood samples taken from the jugular vein were measured on the morning of feeding on day 14 of each period. Dry matter intake, milk protein, and lactose content of cows fed the MCFA-Ca diet were decreased compared with controls, but with no change in milk yield. Plasma ghrelin concentrations were higher in cows fed the MCFA-Ca diet; however, no significant effect was found on glucagon-like peptide-1 concentrations in plasma. Plasma insulin concentrations decreased, but plasma glucagon concentrations remained unchanged in cows fed the MCFA-Ca diet. The concentrations of nonesterified FAs, total cholesterol, and ß-hydroxybutyrate in plasma increased in these cows. In conclusion, dietary MCFAs increase the plasma ghrelin concentrations in lactating dairy cows.

Energy metabolism by splanchnic tissues of mature sheep fed varying levels of lucerne hay cubes.

The objective of this study was to determine the pattern of energy metabolites net flux across the portal-drained viscera (PDV) and total splanchnic tissues (TSP) in mature sheep fed varying levels of lucerne hay cubes. Four Suffolk mature sheep (61.4 ± 3.6 kg BW) surgically fitted with multi-catheters were fed four levels of dry matter intake (DMI) of lucerne hay cubes ranging from 0.4- to 1.6-fold the metabolizable energy (ME) requirements for maintenance. Six sets of blood samples were simultaneously collected from arterial and venous catheters at 30-min intervals. With increasing DMI, apparent total tract digestibility increased linearly and quadratically for dry matter (P < 0.05), quadratically (P < 0.05) with a linear tendency (P < 0.1) for organic matter and tended to increase quadratically (P < 0.1) for NDF. PDV release of volatile fatty acids (VFA) and ß-hydroxybutyric acid was relatively low at 0.4 M and then linearly increased (P < 0.05) with increasing DMI. Net PDV flux of non-esterified fatty acids showed curvilinear decrease from 0.4 to 1.2 M and then increased at 1.6 M. The respective proportions of each VFA appearing in the portal blood differed (P < 0.05) with DMI and this difference was more obvious from 0.4 to 0.8 M than from 0.8 to 1.6 M. Heat production, as a percentage of ME intake (MEI), decreased linearly (P < 0.05) with increasing DMI accounting for 37%, 21%, 16% and 13% for PDV and 62%, 49%, 33% and 27% for TSP at 0.4, 0.8, 1.2 and 1.6 M, respectively. As a proportion of MEI, total energy recovery including heat production, decreased linearly with increasing DMI (P < 0.05) accounting for 113%, 83%, 62% and 57% for PDV and 140%, 129%, 86% and 83% for TSP at 0.4, 0.8, 1.2 and 1.6 M, respectively. Regression analysis revealed a linear response between MEI (MJ/day per kg BW) and total energy release (MJ/day per kg BW) across the PDV and TSP, respectively. However, respective contributions of energy metabolites to net energy release across the PDV and TSP were highly variable among treatments and did not follow the same pattern of changes in DMI.

Effects of forage intake level on nitrogen net flux by portal-drained viscera of mature sheep with abomasal infusion of an amino acid mixture.

This study aimed to investigate the pattern of nitrogen (N) metabolites flux across the portal-drained viscera (PDV) of mature sheep over a wide range of forage intake, and to determine the effect of dry matter intake (DMI) on the PDV recovery of an abomasally infused amino acids (AA) mixture. Four Suffolk mature sheep (61.4 ± 3.6 kg BW) surgically fitted with abomasal cannulae and multi-catheters were fed four levels of DMI of lucerne hay cubes ranging from 0.4 to 1.6 fold the metabolizable energy requirements for maintenance. Each period lasted for 17 days: 7 days for diet adaptation, 5 days for measurement of N balance and N metabolites flux under basal pre-infusion conditions (basal phase) and 5 days for determining the recovery of the infused AA (584 mmol/day) across the PDV (infusion phase). Six sets of blood samples were collected on the last day of both basal and infusion phases. Increasing DMI increased portal release of AA and enhanced N retention. At 0.4 M and as a proportion of digested N, there was a marked drop in total AA-N release accompanied by greater ammonia-N release and urea-N uptake across the PDV. The incremental recovery ratio of infused AA across the PDV was altered with increasing DMI accounting for 0.88, 1.12, 1.23 and 1.31 at 0.4, 0.8, 1.2 and 1.6 M, respectively. In addition, across the individual AA, the net portal recovery ratio of infused methionine and valine increased linearly (P < 0.05) while that of phenylalanine, branched-chain AA and total essential AA tended to increase linearly (P < 0.10) with increasing DMI. These results indicated that DMI affects the net portal recovery of AA available in the small intestine of mature sheep.

Effects of glucose and volatile fatty acids on blood ghrelin concentrations in calves before and after weaning.

The effects of feeding and intravenous injections of glucose and VFA on blood ghrelin concentrations were investigated in calves before and after weaning. Eight Holstein bull calves were fed whole milk, allowed free access to solid feeds, and weaned at 7 wk. Measurements were carried out at 2, 4, 6, 9, 11, and 13 wk, at which time jugular blood samples were taken from 4 calves through a catheter from 10 min before to 120 min after their morning feed at 10 min intervals (Exp. 1). An additional 4 calves of the same age were intravenously injected with glucose (1.0 mmol·kg BW(-1)) and a solution of VFA (2.4 mmol·kg BW(-1), acetate:propionate:butyrate in a 6:3:1 ratio) using a catheter, and jugular blood samples were taken temporally relative to the injection time (Exp. 2). In Exp. 1, blood ghrelin concentrations decreased (P < 0.05) after feeding at all ages. However, preprandial ghrelin concentrations were less (P = 0.025) and the degree of postprandial depression of ghrelin tended to be greater during the postweaning period (P = 0.084) than during the preweaning period. Blood glucose concentrations increased after feeding during the preweaning period (P < 0.05), whereas blood acetate concentrations increased during the postweaning period (P < 0.05). In Exp. 2, injection of VFA induced a greater decrease in blood ghrelin concentrations than glucose injection throughout the entire period (P < 0.05). These results indicate that weaning reduces the basal concentration of blood ghrelin because the circulating VFA derived from ruminal fermentation may more strongly depress blood ghrelin concentrations than glucose.

Plasma concentrations and effects of glucagon-like peptide-1 (7-36) amide in calves before and after weaning.

Glucagon-like peptide-1 (7-36) amide (GLP-1), secreted by the small intestine, has insulinotropic and glucose-lowering action. Basal plasma GLP-1 concentrations were measured in calves around the weaning period, the effect of short-chain fatty acids (SCFA) on plasma GLP-1 concentrations was examined, and the effects of GLP-1 administration on plasma insulin, glucagon, and glucose concentrations were measured. Thirteen Holstein bull calves were fed whole milk and solid feed and weaned at 7 wk of age. Preprandial plasma samples were obtained from 5 calves once a week from week 0 to 13 to measure basal concentrations of plasma GLP-1 and insulin (experiment 1). Four calves were intravenously administered with a mixed solution of SCFA (2.4 mmol/kg body weight [BW]) in week 2 and 11 to measure plasma GLP-1 concentrations (experiment 2). Another 4 calves were intravenously injected with GLP-1 (1.0 µg/kg BW) to elucidate the response of plasma insulin, glucagon, and glucose concentrations in week 1, 2, 4, 6, 7, 9, 11, and 13 (experiment 3). In experiment 1, age and weaning did not affect preprandial basal concentrations of plasma GLP-1 throughout the experimental period. Preprandial insulin concentrations increased after weaning (P < 0.05), and GLP-1 and insulin were more strongly correlated postweaning than preweaning. In experiment 2, intravenous treatment with SCFA increased plasma GLP-1 concentrations in both week 2 and 11 (P < 0.05.) In experiment 3, intravenous GLP-1 treatment decreased plasma glucose concentrations throughout the experiment (P < 0.05), but increased plasma insulin concentrations only after weaning (P < 0.05). Treatment with GLP-1 did not affect plasma glucagon concentrations, regardless of age. These results indicate that preprandial basal concentrations of plasma GLP-1 in calves are not changed by weaning, but SCFA stimulate GLP-1 secretion. The insulinotropic action of GLP-1 is detected only after weaning, but the glucose-lowering action of GLP-1 is not affected by weaning.

Effects of calcium salts of long-chain fatty acids and rumen-protected methionine on plasma concentrations of ghrelin, glucagon-like peptide-1 (7 to 36) amide and pancreatic hormones in lactating cows.

Our objective was to determine the effects of calcium salts of long-chain fatty acids (CLFAs) and rumen-protected methionine (RPM) on plasma concentrations of ghrelin, glucagon-like peptide-1 (7 to 36) amide, and pancreatic hormones in lactating cows. Four Holstein cows in midlactation were used in a 4 by 4 Latin square experiment in each 2-wk period. Cows were fed corn silage-based diets with supplements of CLFAs (1.5% added on dry matter basis), RPM (20 g/d), CLFAs plus RPM, and without supplement. Jugular blood samples were taken from 1 h before to 2 h after morning feeding at 10-min intervals on day 12 of each period. CLFAs decreased dry matter intake, but RPM did not affect dry matter intake. Both supplements of CLFAs and RPM did not affect metabolizable energy intake and milk yield and composition. Plasma concentrations of NEFAs, triglyceride (TG), and total cholesterol (T-Cho) were increased with CLFAs alone, but increases of plasma concentrations of TG and T-Cho were moderated by CLFAs plus RPM. Calcium salts of long-chain fatty acids increased plasma ghrelin concentration, and the ghrelin concentration with CLFAs plus RPM was the highest among the treatments. Plasma concentrations of glucagon-like peptide-1, glucagon, and insulin were decreased with CLFAs, whereas adding RPM moderated the decrease of plasma glucagon concentration by CLFAs. These results indicate that the addition of methionine to cows given CLFAs increases plasma concentrations of ghrelin and glucagon associated with the decrease in plasma concentrations of TG and T-Cho.

Plasma ghrelin concentration is decreased by short chain fatty acids in wethers.

To examine the effects of short chain fatty acids (SCFAs) on plasma ghrelin concentration, 4 wethers were injected intravenously with SCFA solutions [acetate (ACE), propionate (PRO), and butyrate (BUT) (0.8 mmol/kg BW)] and saline. The experiment was conducted after a 4 × 4 Latin square design. Each solution was injected into the jugular vein catheter with blood samples taken at -10, 0, 5, 10, 15, 20, 25, 30, 40, 50, and 60 min relative to the injection time also from this catheter. Plasma ghrelin concentrations decreased after injection with ACE, PRO, and BUT. Although plasma glucose concentrations increased after injection with PRO and BUT (P < 0.05), the increment areas were greater with BUT than with PRO. Plasma insulin concentrations increased after injection with PRO and BUT (P < 0.05). The decrement areas in plasma ghrelin concentrations were equal in ACE, PRO, and BUT. These data suggest that SCFAs inhibit ghrelin secretion in wethers and not through increased circulating glucose and insulin as previously proposed.

Effect of amino acid and glucose administration following exercise on the turnover of muscle protein in the hindlimb femoral region of thoroughbreds.

REASONS FOR PERFORMING STUDY: In man, muscle protein synthesis is accelerated by administering amino acids (AA) and glucose (Glu), because increased availability of amino acids and increased insulin secretion, is known to have a protein anabolic effect. However, in the horse, the effect on muscle hypertrophy of such nutrition management following exercise is unknown. OBJECTIVES: To determine the effect of AA and Glu administration following exercise on muscle protein turnover in horses. We hypothesise that administration of AA and Glu after exercise effects muscle hypertrophy in horses, as already shown in man and other animals. METHODS: Measurements of the rate of synthesis (Rs) and rate of degradation (Rd) of muscle protein in the hindlimb femoral region of thoroughbred horses were conducted using the isotope dilution method to assess the differences between the artery and iliac vein. Six adult Thoroughbreds received a continuous infusion of L-[ring-2H5]- phenylalanine during the study, the stable period for plasma isotope concentrations (60 min), resting periods (60 min), treadmill exercise (15 min) and recovery period (240 min). All horses were given 4 solutions (saline [Cont], 10% AA [10-AA], 10% Glu [10-Glu] and a mixture with 10% AA and 10% Glu [10-Mix]) over 120 min after exercise, and the Rs and Rd of muscle protein in the hindlimb measured. RESULTS: The average Rs during the 75-120 min following administration of 10-Mix was significantly greater than for the other solutions (P<0.05). The second most effective solution was 10-AA, and there was no change in Rs after 10-Glu. CONCLUSIONS: Administration of AA following exercise accelerated Rs in the hindlimb femoral region, and this effect was enhanced when combined with glucose, because of increasing insulin secretion or a decreased requirement for AA for energy. POTENTIAL RELEVANCE: Further studies are required regarding the effect on muscle hypertrophy of supplementing amino acids and glucose in the feed of exercising horses.

Glutamine metabolism in ovine splanchnic tissues: effects of infusion of ammonium bicarbonate or amino acids into the abomasum.

This study investigates the effects of increased NH3 or amino acid supply on glutamine utilisation and production by the splanchnic tissues of fed sheep. Six sheep, prepared with vascular catheters in the aorta, mesenteric, portal and hepatic veins, were fed grass pellets to 1.1 x energy maintenance requirements. Each treatment involved a 4 d abomasal infusion, of either ammonium bicarbonate (AMM; 234 micromol/kg(0.75 per min), water (CONT), or a mixture of amino acids that excluded glutamine and glutamate (AA; 46.8 micromol amino acid-N/kg(0.75) per min). The treatments simulated nutritional extremes in terms of the balance of absorbed N. Kinetics across the whole gut and the liver were monitored during an intra-jugular infusion of [5-(15N)]glutamine. Blood flow across the whole gut or liver were unaffected by treatment. Both AMM and AA infusions doubled the hepatic release of urea-N compared with CONT (P<0.02). AA infusion decreased arterial glutamine concentration by 26% (P<0.01) and 23 % (P<0.05) compared with AMM and CONT respectively. Despite this, whole-body glutamine flux was not affected by treatment. In contrast, AMM infusion increased hepatic glutamine production by 40% compared with CONT (P<0.02). This provided a mechanism to ensure NH3 supply to the periphery was maintained within the normal low physiological levels. Hepatic glutamine utilisation tended to increase during AA infusion, probably to ensure equal inflows of N to the ornithine cycle. Between 6 and 10% of NH3 absorbed across the digestive tract was derived from the amido-N of glutamine. Overall, splanchnic glutamine utilisation accounted for 45-70% of whole-body glutamine flux.

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.

Transfers of N metabolites across the ovine liver in response to short-term infusions of an amino acid mixture into the mesenteric vein.

The effect of acute (4.5 h) infusions into the mesenteric vein of an amino acid (AA) mixture, which simulated the composition of rumen microbial protein, on net transfers of NH3, urea and total AA across the portal-drained viscera (PDV) and liver in the ovine has been examined. Four wether sheep were surgically prepared with vascular catheters across the PDV and liver (Lobley et al. 1995) and were offered a basal diet of 1000 g grass pellets/d (approximately 1.4 x energy maintenance). Each animal was infused at weekly intervals with one of four dilutions of the AA mixture. These dilutions provided 0.44, 0.88, 1.32 and 1.84 mmol AA-N/min infused, the lowest of which approximately doubled the net absorption of AA-N from the basal diet. Animals were treated with heparin to allow continuous collection of blood by peristaltic pump for 2 h preceding, and between 0.5-2.5 and 2.5-4.5 h after, the start of the AA infusions. Blood flow in the hepatic artery increased (100 v. 208 g/min; P = 0.002) in response to AA infusion, while hepatic portal venous flow decreased (2090 v. 1854 g/min; P = 0.006). The AA infusion also stimulated O2 uptake by the PDV (P < 0.001) and liver (P = 0.016). Absorption across the PDV and hepatic removal of NH3 were unchanged between basal and amino acid infusion conditions. Urea-N removal across the PDV was unaltered, but hepatic production increased (P < 0.001) with level of AA infusion. During infusions, net appearance of AA across the PDV was below the theoretical level. This may have been due to inhibition of AA uptake from the small intestine, and/or increased removal by the digestive tract of AA from the systemic circulation associated with greater arterial concentrations. Hepatic extraction of AA increased with level of infusion, both for total AA and those included in the infusate. Total hepatic urea-N production tended towards a maximum (estimated as 2 mumol N/g liver wet weight per min). The AA removed by the liver and not used for ureagenesis remained similar (170 mumol AA-N/min) between basal and AA infusions. This was presumed available for anabolic purposes (mainly synthesis of export proteins). The proportion of net AA-N appearance (absorption plus infused) across the PDV removed by the liver declined from 0.71 to 0.53 between basal and AA infusions. In contrast to findings from cattle (Wray-Cahen et al. 1997), increased AA infusion did not alter the net removal of glutamine across the liver. This may reflect differences between the studies in NH3: AA-N absorbed. Further differences between the cattle study and the current findings may relate to the different physiological state (pregnancy v. growth), which may alter the partition of AA between anabolic and catabolic fates.