Histidine optimal supply in dairy cows through determination of a threshold efficiency.

Two His deletion studies were conducted to examine the mechanisms used by dairy cows to support milk true protein yield (MTPY) when His supply is altered. The potential mechanisms involved in how the efficiency of utilization of His varied included reduced catabolism, more efficient mammary usage, and use of His labile pools. For the first study, 5 multicatheterized cows were used in a 4 × 4 Latin square plus 1 cow with 14-d periods. Treatments were abomasal infusion of increasing doses of His (0, 7.6, 15.2, and 20.8 g/d) in addition to a mixture of AA (595 g/d; casein profile excluding His). Cows were fed the same protein-deficient diet throughout the study. The MTPY increased linearly with a quadratic tendency with increasing doses of His. Muscle concentrations of carnosine, a His-based dipeptide, tended to increase in a quadratic manner with increasing His supply, suggesting that the 0- and 7.6-g doses were insufficient to cover His requirement. Liver catabolism of His decreased as His supply decreased. Mammary fractional removal of His was considerably greater at low His supply, but the ratio of His mammary net uptake to milk output was not affected by the rate of His infusion, averaging 1.02. The mechanisms to face a reduced His supply included reduced His hepatic catabolism, more efficient His mammary use of lowered arterial supply, and, to a lesser extent, use of His labile pools. Two independent estimates of His efficiency were calculated, one based on the sum of exported proteins (measured MTPY plus estimated metabolic fecal protein and scurf; i.e., the anabolic component, EffMTPY) and the other based on liver removal (i.e., the catabolic component). These 2 estimates followed the same pattern of response to His supply, decreasing with increasing His supply. The EffMTPY at which MTPY peaked was 0.785. For the second study, 6 cows were used in a 6 × 6 Latin square with 7-d periods. Two greater doses of His (30.4 and 38.0 g/d) were added; otherwise, the nutritional design was similar to the first study. In this second study, the indicator AA oxidation technique was used instead of the multiorgan approach, with labeled Leu as the indicator of His utilization. The MTPY peaked and Leu oxidation reached the nadir at an average EffMTPY of 0.763. Combined across both studies, the data indicate that optimal usage of His would occur at a threshold EffMTPY of 0.77. The agreement between experimental approaches across both studies indicates that the biological optimal supply of His expressed in grams per day could be calculated as the sum of exported proteins divided by this EffMTPY plus estimated endogenous urinary excretion.

Dietary carbohydrate rather than protein intake drives colonic microbial fermentation during weight loss.

PURPOSE: High protein weight loss diets are effective in aiding body weight management. However, high protein and low carbohydrate intakes can alter colonic fermentation profiles in humans and may impact on colonic health. This study aims to identify the most important dietary contributors to colonic fermentation during diet-controlled weight loss. METHODS: Overweight or obese male volunteers (n = 18) consumed a body weight maintenance diet (fed at 1.5× basic metabolic rate, BMR) followed by three weight loss diets (fed at 1× BMR) for 10 days each in a cross-over design. Weight loss diets were designed as normal protein (NPWL, 15% of energy from protein, 55% from carbohydrate), normal protein enriched with free amino acids and moderate amounts of carbohydrate (NPAAWL, 15% of energy from protein, 15% from free AA, 40% from carbohydrate) or high protein containing moderate amounts of carbohydrate (HPWL, 30% of energy from protein, 40% from carbohydrate). Faecal samples collected at the end of each diet period were profiled for dietary metabolites using LC-MS/MS. RESULTS: This study shows that the NPWL diet only induced very minor changes in the faecal metabolome, whereas NPAAWL and HPWL diets decreased carbohydrate-related metabolites (butyrate, ferulic acid) and increased protein-related metabolites. Most faecal metabolites were correlated with dietary carbohydrate and not protein intake. CONCLUSION: This study demonstrates that dietary carbohydrate is the main driver of colonic fermentation in humans and that a balance between dietary carbohydrate and protein should be maintained when designing safe, effective and healthy weight loss diets.

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.

Circulating amino acids in blood plasma during the peripartal period in dairy cows with different liver functionality index.

The liver functionality index (LFI) measures the changes of albumin, cholesterol, and bilirubin concentrations between 3 and 28 d postpartum. This composite index, based on variables with direct relevance to liver-specific plasma protein synthesis (albumin), hepatic/intestinal lipoprotein synthesis (cholesterol), and clearance of breakdown products of heme catabolism (bilirubin), provides a tool for evaluating manifestations of hepatic disease. Both energy and protein metabolism are likely to be affected by various physiological challenges in this period but have not been tested systematically. The present study was conducted to profile AA in cows with high or low LFI during the peripartal period and relate this to production outcomes. Eighteen multiparous cows were used from -21 through 28 d around parturition and divided retrospectively into the high or low LFI group. Blood samples were obtained on -21, -14, -7, 1, 3, 7, 10, 14, 17, 21, and 28 d relative to calving, and biomarkers and AA in plasma were measured. Grouping based on LFI resulted in 8 cows with high LFI (HLFI) and 10 cows with low LFI (LLFI). Although the temporal response in dry matter intake (DMI, 16.3 kg/d) and body condition score (2.56) did not differ, cows with high compared with low LFI had greater overall milk production (37.9 vs. 32.9 kg/d) although energy-corrected milk yield did not differ (42.6 vs. 38.7 kg/d). As expected, cows grouped as LLFI had lower cholesterol and albumin but greater bilirubin after calving compared with HLFI animals. Despite similar temporal responses in DMI between groups, concentrations of total AA were greater in HLFI, particularly after calving. Although concentrations of total essential AA (EAA) and branched-chain AA did not differ with LFI status, cows in HLFI had greater concentrations of Thr and Ile postpartum. Nearly all plasma AA concentrations followed the general trend of a nadir at 1d after calving followed by a gradual increase to prepartal levels before 28 d. Glycine was the only AA exhibiting a gradual increase in concentration through the transition, with a maximum at 7d postpartum followed by a gradual decrease. We detected no effect of LFI status on plasma Lys, which decreased markedly from -21d to calving, followed by an increase to prepartal values by d7. In contrast, concentrations of Met and His decreased markedly between -21 and 10d and did not reach prepartal values by 28 d. The marked decrease in Gln concentration after calving regardless of LFI might compromise immune function during this period. Overall, the results indicate the existence of an association among inflammation, liver function postpartum, and AA plasma concentrations, irrespective of temporal differences in DMI. Cows with better indices of liver function produced more milk and maintained greater concentrations of total AA and some EAA such as Thr and Ile. Whether these AA played a direct role in the greater milk production remains to be determined.

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.

A randomized crossover study to assess the effect of an oat-rich diet on glycaemic control, plasma lipids and postprandial glycaemia, inflammation and oxidative stress in Type 2 diabetes.

AIMS: In the UK, lifestyle intervention is first-line management in Type 2 diabetes. It is unclear what type of diet is most efficacious for improving glycaemic control. This study investigated the effects of an oat-enriched diet on glycaemic control, postprandial glycaemia, inflammation and oxidative stress compared with standard dietary advice. METHODS: In a randomized crossover design, 27 volunteers with Type 2 diabetes, managed on diet and lifestyle only, were observed for two consecutive 8-week periods following either the oat-enriched diet or re-enforced standard dietary advice. Volunteers attended at baseline (habitual intake) and 8 and 16 weeks. Measurements included basic clinical measurements and fasted and postprandial (3-h) glucose and insulin in response to a healthy test meal. Markers of inflammation and oxidative stress, including high-sensitivity C-reactive protein, interleukin 6, interleukin 18, tumour necrosis factor-alpha, adiponectin, thiobarbituric acid reactive substances, oxygen radical antioxidant capacity, oxidized LDL and urinary isoprostanes, were also measured at fasting and in the postprandial period. RESULTS: There were no diet-related effects on glycaemic control or glycaemic or insulinaemic responses to the test meal. Total cholesterol (5.1 ± 1.0 vs. 4.9 ± 0.8 mmol/l, P = 0.019) concentrations declined following the oat-enriched diet compared with standard dietary advice. There was a postprandial decline in adiponectin concentration (P = 0.009), but no effect of dietary intervention. None of the measures of oxidative stress or inflammation were altered by the oat-enriched diet compared with standard dietary advice. CONCLUSION: The oat-enriched diet had a modest impact on lipid lowering, but did not impact on oxidative stress or inflammation in these volunteers with Type 2 diabetes.

Triennial Lactation Symposium: Mammary metabolism of amino acids in dairy cows.

Although in dairy cows the mammary gland (MG) is the major net user of essential AA (EAA) supply, milk protein synthesis from absorbed EAA is not a straightforward process. Early studies identified 2 groups of EAA based on different pattern of mammary utilization: group 1 [Met, Phe (+Tyr), Trp], where MG uptake was similar to secretion in milk protein, and group 2 (Arg, Ile, Leu, Lys, Thr, and Val), where uptake exceeded milk protein output. This review examines the validity of this classification under variable protein supply through a meta-analysis, with the outcomes then explained with studies in which the fates of individual EAA were monitored using isotope approaches. For the meta-analysis, the Fick principle, based on stoichiometric transfer of Phe+Tyr uptake to milk protein, was used to estimate mammary plasma flow across all studies. This approach was judged acceptable because doubling Phe supply did not result in mammary oxidation of Phe+Tyr and either limited or no contribution of peptides to Phe and Tyr mammary supply could be detected. The AA content of proteins synthesized by the MG was estimated from milk protein composition, and the uptake-to-output ratio (U:O) for individual AA was re-calculated based on these assumptions. Analysis of individual samples by isotopic dilution resulted in reduced variance compared with analysis on pooled samples performed with an AA analyzer. Globally, the U:O of His and Met is maintained close to unity under variable protein supply. The group 2 AA could be subdivided. First, the U:O for group "2v" AA (Ile, Leu, Val, and Lys) is greater than 1 and varied with protein supply. Accordingly, the increased U:O of Leu, induced by duodenal casein infusion, led to extra-mammary Leu oxidation. Decreasing Lys supply decreased Lys U:O and the associated transfer of N to non-EAA, mainly to Glx, Asx, Ser, and Ala. Second, the U:O of group "2nv" AA, Arg and Thr, does not vary with protein supply. The Arg U:O averages 2.5, whereas the Thr U:O, albeit averaging 1.2, does not differ from unity. Excess of both these AA is probably directed toward the synthesis of non-EAA rather than energy supply. Overall, the ability of the MG to use excess EAA-N supply offers alternative sources of N and C for energy provision, lactose synthesis and non-EAA synthesis. The latter function spares dietary non-EAA for other necessary processes, such as gluconeogenesis and energy supply, in other tissues to support lactation.

Is D-methionine bioavailable to the dairy cow?

Rumen-protected forms of Met contain an equimolar mixture of the D- and L-isomers. Only L-Met can be directly used for protein synthesis, but it is unclear how much of the D-isomer can be transformed into L-Met in ruminants. Four lactating dairy cows, with an average milk yield of 32.4 kg/d, received a basal diet (12.5% crude protein, supplying 1,718 g/d of metabolizable protein) in 12 equal meals per day plus an abomasal infusion of amino acids (590 g/d, casein profile without Met). They were used in 3 consecutive studies to determine utilization of D-Met. First, the cows each received portal vein infusions for d of 5, 10, or 15 g/d of DL-Met in a Youden square. On the last day of each period, 6 arterial samples were collected at 45-min intervals. Concentrations of L- and D-Met were determined on a chiral column by gas chromatography-mass spectrometry. Portal infusion of 5, 10, and 15 g/d of DL-Met increased plasma total Met concentrations (19.7, 25.0, and 34.4±0.6 µM) and the proportion of Met as D (19.4, 30.5, and 37.3±0.7%). The fractional removal of D-Met was 6 to 7 times lower than the fractional removal of L-Met, with mean half-lives of 52 versus 8 min, respectively. Second, the same cows were infused for 8 h with L[methyl-(2)H(3)]Met at 1.3 mmol/h; at 2 h, cows received a bolus injection i.v. of D-[1-(13)C]Met (6.8 mmol), and arterial samples were collected after 10, 20, 30, 40, 60, 90, 120, 150, 180, 240, 300, 360, 420, and 480 min. Expressed relative to L-[(12)C]Met; that is, as tracer:tracee ratios, enrichments of plasma D-[1-(13)C]Met and L-[1-(13)C]Met averaged 1.77±0.14 and 0.144±0.026, respectively, 10 min after the bolus injection and declined exponentially thereafter. A minimum of 75±3% of the D-[1-(13)C]Met was transformed into L-[1-(13)C]Met. Third, the cows received, in a crossover design, an abomasal infusion for D of either DL-Met or L-Met (1g/d) and, on the last day of each experimental period, blood samples were collected simultaneously from arterial, portal, hepatic, and mammary vessels. Arterial total Met concentrations were higher with DL- versus L-Met infusions (37.4 vs. 25.4±0.5 µM), with 37.1±5.0% as D-Met. The mammary gland did not extract any D-Met. Hepatic removal of D-Met was observed, but was numerically lower than the fractional extraction of L-Met. In conclusion, much of the D-Met is transformed into L-Met by the dairy cow but at a slow rate. No uptake of D-Met occurs across the mammary gland but L-Met synthesized from the D-isomer elsewhere in the body can be utilized for milk protein synthesis.

Metabolism of 2-hydroxy-4-(methylthio)butanoate (HMTBA) in lactating dairy cows.

The objectives of the current study were to determine the fate and contribution to Met kinetics of 2-hydroxy-4-(methylthio)butanoate (HMTBA) at the whole-body, splanchnic, and mammary levels. Four multicatheterized cows (31.3 kg of milk/d; 17.7 kg of DMI/d) were used in a crossover design, with two 1-wk periods, to determine the metabolic fate of HMTBA and its effect on Met metabolism. Over the last 2 d of each period, cows were infused, via a jugular vein, with saline or HMTBA (Alimet, Novus International Inc., St. Louis, Mo) at the rate of 36 g/d. During the last 8h, the HMTBA infusion was substituted by equimolar [1-(13)C]HMTBA (8.79 mmol/h) and l[methyl-(2)H(3)]Met (1.31 mmol/h) was infused in all cows. During the last 5h, hourly samples (n=6) were collected to determine plasma flows plus the isotopic enrichments (IE) and concentrations of HMTBA ((13)C) and Met (both (13)C and (2)H(3)) in plasma from an artery plus portal, hepatic, and mammary veins. The IE of [(13)C] and [(2)H(3)]Met were also determined in milk protein taken over the last 1h of infusion in HMTBA-infused cows. The infused HMTBA increased whole-body plasma flux of Met by 6.5 mmol/h (from 17.9 to 24.4 mmol/h). Based on enrichments of (13)C-labeled Met, 3.8 mmol/h of Met flow through plasma was derived directly from HMTBA. These 2 estimates accounted for between 43 to 74% of the HMTBA dose infused, contributing to increased whole-body Met availability. Although the portal-drained viscera, liver, and mammary gland (MG) extracted 11, 37, and 3.4%, respectively, of the infused HMTBA, tissue net Met fluxes were either unchanged (portal-drained viscera, MG) or even reduced (liver: -7.9 vs. -2.4±0.6 mmol/h). Therefore, net postsplanchnic supply of Met decreased from 7.0 to 2.9 mmol/h between control and HMTBA-infused cows, compared with needs for milk protein secretion of 7.6 and 8.1 mmol/h, respectively. The HMTBA provided directly 15% of the Met required for milk protein secretion, with 0.2 mmol/h synthesized within the MG, whereas 1.1 mmol/h originated from Met produced in other tissues and transported to the MG through blood circulation. Most of the remainder needed by the MG arose from unlabeled Met released from protein breakdown in extra-splanchnic tissues and that was not reused to support intracellular protein synthesis, as this function was performed by Met synthesized from HMTBA in situ. Absorbed HMTBA, therefore, both produces and spares Met for use by the MG.

Effect of litter size and bacitracin administration on tissue protein synthesis of lactating rabbit does.

Bacitracin is an antibiotic used in rabbit husbandry to control microbial digestive pathologies. Collateral effects on absorption and mucosal development have been reported and these may impact on protein metabolism. This study aims to analyse the effect of the antibiotic on protein synthesis in lactating does because mammary gland metabolism and milk output should provide a sensitive index of any undesirable action of bacitracin. Rates of protein synthesis were measured in mammary gland, liver, intestinal mucosa and muscle of lactating rabbits does by injecting a flooding dose of [(2)H(5)]phenylalanine into the auricular artery of two groups (each n = 8) of New Zealand White does fed different experimental diets. The control group (C) received the basal diet and the bacitracin group (B) ingested the same diet but supplemented with bacitracin (100 mg/kg). Animals received the experimental diet from day 28 of pregnancy until day 26 of lactation when they were slaughtered. Just after birth, litter size was adjusted by cross-fostering either to five or nine pups (four does per dietary treatment). The relative weight of the liver tended to be greater in those females receiving the B diet (27 v. 22.5 g/kg BW; P < 0.07), while diet did not affect mammary gland weight (255.7 ± 10.59 g). Fractional protein synthesis rate (FSR) was higher for intestinal mucosa (duodenum; 51.7% ± 2.09%/day) followed by mammary gland and liver (38.29 ± 2.62%/day and 40.2 ± 1.98%/day, respectively), and the lowest value was observed in muscle (2.92 ± 0.26%/day; P < 0.0001). Bacitracin treatment lowered FSR in the mammary gland by 23% (P = 0.024) and this was independent of litter size. Conversely, FSR in the duodenum was not affected by antibiotic treatment but reduced by 15% (P = 0.021) for the larger litter size.

Effect of method of conservation of timothy on endogenous nitrogen flows in lactating dairy cows.

The effect of the method of conservation of forage on endogenous N (EN) secretion was studied using a 15N isotope dilution technique in 4 lactating Holstein cows selected from a replicated 3x3 Latin square. Cows were equipped with ruminal, duodenal (n=4), and ileal (n=2) cannulas. Diets comprised 44% concentrate plus first-cut timothy conserved either as hay or as restrictively (formic) or extensively (inoc) fermented silage. Crude protein contents of hay, formic, and inoc averaged 10.4, 13.6, and 14.8%, respectively. Total EN flow and free EN at the duodenum were increased with hay compared with silages but were similar when expressed as proportion of duodenal N flow, with total EN flow averaging 25.8, 23.9, and 23.9% for hay, formic, and inoc, respectively, and free EN at the duodenum averaging 11.5, 9.8, and 9.7% for hay, formic, and inoc, respectively. Flow of bacterial N at the duodenum originating from an endogenous source tended to be higher with inoc compared with formic. Overall, the proportion of bacterial N derived from endogenous sources and urea was similar between treatments, averaging 23 and 15%, respectively. In the feces, flow of EN was similar across treatments and averaged 31% of total fecal N. More than 70% of fecal EN originated from undigested secretions into the forestomach. Absorption of N from the forestomach tended to increase for silages compared with hay. In conclusion, EN represented an important fraction of N flowing at the duodenum and in the feces. The free EN and the total EN at the duodenum were altered by the different methods of forage conservation studied. Estimation of true dietary N supply and requirements of the dairy cow should allow for endogenous N flows and losses.

Use of the cellular model of body composition to describe changes in body water compartments after total fasting, very low calorie diet and low calorie diet in obese men.

INTRODUCTION: The cellular model of body composition divides the body in body cell mass (BCM), extracellular solids and extracellular fluids. This model has been infrequently applied for the evaluation of weight loss (WL) programmes. OBJECTIVES: (1) To assess changes in body compartments in obese men undergoing fasting, very low calorie diet (VLCD) and low calorie diet (LCD); (2) to evaluate two cellular models for the determination of changes in BCM, fat mass (FM) and body fluids. MATERIALS AND METHODS: Three groups of six, obese men participated in a total fast (F) for 6 days, a VLCD (2.5 MJ per day) for 3 weeks or an LCD (5.2 MJ per day) for 6 weeks. Body composition was measured at baseline and after small ( approximately 5%) and moderate ( approximately 10%) WL. FM was measured using a four-compartment model. Total body water (TBW) and extracellular water (ECW) were, respectively, measured by deuterium and sodium bromide dilution and intracellular water (ICW) calculated by difference. Two cellular models were used to measure BCM, FM and body fluids distribution. RESULTS: After about 5%WL changes in TBW were F=-3.2+/-1.2 kg (P<0.01), VLCD=-1.2+/-0.6 kg (P<0.01), LCD=-0.3+/-0.9 kg(n.s.). The contribution of TBW to total body mass loss was indirectly associated with FM loss. ECW increased during fasting (+1.5+/-3.1 kg, n.s.), decreased during the VLCD (-2.0+/-1.5 kg, P<0.05) and remained unchanged at the end of the LCD (-0.3+/-1.6 kg, n.s.). ICW significantly decreased during fasting (-4.7+/-3.9 kg, P<0.05) but did not change in the LCD and VLCD groups. The loss of BCM was more significant in the fasting group and it was directly associated with changes in ICW. CONCLUSIONS: After a 6-day period of fasting we observed more ICW losses and less fat mobilization compared with VLCD and LCD. The cellular model of body composition is suitable for the characterization of changes in body fluids distribution during WL.

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

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

Differences in splanchnic metabolism between late gestation and early lactation dairy cows.

In the transition from the pre- to postcalving state, the demands on the cow increase from support of gestation to high rates of milk production. This extra demand is met partly by increased intake but may also involve altered metabolism of major nutrients. Six multiparous Holstein cows were used to monitor changes in net fluxes of nutrients across the portal-drained viscera and liver (splanchnic tissues) between late gestation and early lactation. Blood samples were obtained simultaneously from the portal, hepatic, and subcutaneous abdominal veins and the caudal aorta 18 d before expected calving and 21 or 42 d after calving. On the day of blood sampling and the 3 d preceding sampling, cows were fed every 2 h. The precalving (1.63 Mcal of net energy for lactation/kg and 1,326 g of metabolizable protein/d) and postcalving (1.72 Mcal of net energy for lactation/kg and 2,136 g of metabolizable protein/d) diets were based on corn silage, alfalfa hay, and corn grain. Dry matter intake increased postcalving. Net splanchnic release of glucose increased postpartum because of tendencies for both increased portal absorption and net liver release. Increased removal of lactate, rather than AA, contributed to the additional hepatic gluconeogenesis. Although portal absorption of AA increased with intake at the onset of lactation, hepatic removal of total AA-N tended to decline. This clearly indicates that liver removal of AA is not linked to portal absorption. Furthermore, net liver removal relative to total liver inflow even decreased for Gly, His, Met, Phe, and Tyr. Together, these data indicate that in early lactation, metabolic priority is given to direct AA toward milk protein production rather than gluconeogenesis, in cows fed a corn-based ration.

Responses in mammary and splanchnic metabolism to altered lysine supply in dairy cows.

Lysine is usually taken up in excess by the mammary gland (MG) relative to milk protein output, allowing for mammary synthesis of non-essential (NE) amino acids (AA) from Lys-N. It is unclear whether this NEAA synthesis from Lys is obligate or whether more efficient use of Lys can be made under limiting conditions. Six multi-catheterized dairy cows received a basal diet low in protein plus an abomasal infusion of AA (560 g/day) with or without Lys (50.3 g/day), in a crossover design with 7-day periods. On day 7, all cows received a 7.5-h jugular infusion of [2-15N]lysine. Six blood samples were collected from arterial, portal, hepatic and mammary vessels at 45 min intervals. In addition, cows were milked at 6 and 7 h with the milk casein plus arterial and mammary plasma collected at 7 h analyzed for AA enrichment. Milk protein concentration and casein yield tended (P < 0.10) to decrease with Lys deletion, while Lys secretion in milk protein was lowered (P < 0.05). The addition of Lys in the AA mixture increased the net portal absorption of Lys by the amount infused, suggesting limited oxidation of this extra supply by the gut. Net liver flux of Lys was unaltered by treatment and, therefore, net splanchnic release of Lys reflected closely the amounts absorbed. For both treatments, however, post-liver supply was greater than mammary uptake, which exceeded milk output. Nonetheless, while Lys deletion decreased mammary uptake by 10.1 mmol/h, Lys in milk protein secretion was reduced by only 3.9 mmol/h. On a net basis, there was no evidence of the additional uptake of any other measured AA during the Lys deletion. The mammary uptake to output ratio of Lys decreased from 1.37 to 1.12, but still showed an excess with Lys deletion. The total amount of 15N in milk protein did not change with treatment but the distribution into AA was altered. In conditions that simulated normal feeding (Lys infused), 83% of the 15N was present as Lys, with Glx, Asx, Ser and Ala harvesting, respectively, 6.8%, 2.4%, 2.1% and 1.0%. With Lys depletion, N-transfers from Lys to other AA within the MG were still present, but rates were considerably lower. This would suggest that part, at least, of Lys catabolism in the MG is either needed or cannot be prevented completely, even at low supply of Lys. Such catabolism will provide N to support the synthesis of NEAA.

Human colonic microbiota associated with diet, obesity and weight loss.

BACKGROUND: It has been proposed that the development of obesity in humans is influenced by the relative proportions of the two major phyla of bacteria (Bacteroidetes and Firmicutes) present in the large intestine. OBJECTIVE: To examine the relationships between body mass index, weight loss and the major bacterial groups detected in fecal samples. DESIGN: Major groups of fecal bacteria were monitored using fluorescent in situ hybridization (FISH) in obese and non-obese subjects under conditions of weight maintenance, and in obese male volunteers undergoing weight loss on two different reduced carbohydrate weight-loss diets given successively for 4 weeks each. RESULTS: We detected no difference between obese and non-obese individuals in the proportion of Bacteroidetes measured in fecal samples, and no significant change in the percentage of Bacteroidetes in feces from obese subjects on weight loss diets. Significant diet-dependent reductions in a group of butyrate-producing Firmicutes were, however, detected in fecal samples from obese subjects on weight loss diets. CONCLUSIONS: Diets designed to achieve weight loss in obese subjects can significantly alter the species composition of the gut microbiota, but we find no evidence that the proportions of Bacteroidetes and Firmicutes among fecal bacteria have a function in human obesity.

Distribution of 15N in amino acids during 15N-leucine infusion: impact on the estimation of endogenous flows in dairy cows.

The distribution of (15)N in AA during [(15)N]Leu infusion and its impact on the estimation of endogenous nitrogen (EN) flows in dairy cows was evaluated in 4 lactating cows equipped with ruminal, duodenal (n = 4), and ileal (n = 2) cannulae fed a silage-based diet during a 35-d experimental period. To label EN, starting on d 27, an infusion of L-[(15)N]Leu (0.45 mmol/h) was performed for 200 h. Samples of feed, duodenal and ileal digesta, feces, blood, urine, and mucosa of the rumen and duodenum were taken at 0900, 1100, 1300, and 1500 h on d 34 and at 0800, 1000, 1200, and 1400 h on d 35. The enrichment and fluxes of total N and individual AA were determined and used to calculate the EN flows at the duodenum, ileum, and in the feces. Based on the concept that EN comprises desquamation and secretions, EN flows were estimated, using as representative of the enrichment of EN only the enrichment of the gut mucosa (upper limit) or the average of the mucosa and the export protein enrichment (assumed to have a similar enrichment to casein; lower limit). Estimations of duodenal and fecal EN flows using the isotope dilution of (15)N-total and (15)N-Leu were not different and EN was an important fraction of duodenal and fecal flows, representing 14 to 30% of the duodenal flow and 18 to 31% of the fecal flow, depending on the dilution method used. The total EN flow at the duodenum is present in approximately equal proportions as either free EN or EN incorporated into bacterial protein. Ileal EN flow was 18% greater than the fecal EN flow. Using the combination of the gut and export protein, the duodenal and fecal EN flows estimated with the isotopic dilution of Leu vs. other labeled AA were less different than when estimated using the enrichment of gut mucosa alone. The current approaches have highlighted that present prediction schemes probably underestimate EN flows at the duodenum and, in consequence, overestimate net protein and AA supply. Refinement of the procedures may allow direct and accurate estimation of metabolic fecal protein, an important component of the so-called maintenance requirement of dairy cows.

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.

Short communication: Absorption of 2-hydroxy-4-methylthiobutanoate in dairy cows.

The objective of this study was to measure net portal absorption of 2-hydroxy-4-methylthiobutanoate (HMTBA) in dairy cows. Four multicatheterized lactating cows were used in a cross-over design with 7-d experimental periods. They were fed every other hour a total mixed ration and received, in addition, twice a day, 12.5 or 25 g/meal of HMTBA. On the last day of treatment, net portal absorption of HMTBA was numerically greater after the 25-g compared with the 12.5-g bolus meal of HMTBA, and the amount absorbed relative to the dose ingested was unchanged between treatments averaging 11.2 +/- 4.7% of the dose. This represents a minimum value of HMTBA availability because it does not take into account any HMTBA metabolized to Met by gut tissues (in sheep this amounted to another 5% of the dose). A rapid method to estimate net portal absorption based on temporal variations of the peripheral plasma HMTBA concentrations following the HMTBA meal is also presented. Based on the good relationship (concordance correlation coefficient = 0.97) observed between direct measurements and estimations, this simplified approach offers a reasonable approach to assess HMTBA absorption under different feeding situations.