N.B. Kristensen

Gluconeogenesis in dairy cows: the secret of making sweet milk from sour dough.

Gluconeogenesis is a crucial process to support glucose homeostasis when nutritional supply with glucose is insufficient. Because ingested carbohydrates are efficiently fermented to short‐chain fatty acids in the rumen, ruminants are required to meet the largest part of their glucose demand by de novo genesis after weaning. The qualitative difference to nonruminant species is that propionate originating from ruminal metabolism is the major substrate for gluconeogenesis. Disposal of propionate into gluconeogenesis via propionyl‐CoA carboxylase, methylmalonyl‐CoA mutase, and the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK) has a high metabolic priority and continues even if glucose is exogenously supplied. Gluconeogenesis is regulated at the transcriptional and several posttranscriptional levels and is under hormonal control (primarily insulin, glucagon, and growth hormone). Transcriptional regulation is relevant for regulating precursor entry into gluconeogenesis (propionate, alanine and other amino acids, lactate, and glycerol). Promoters of the bovine pyruvate carboxylase (PC) and PEPCK genes are directly controlled by metabolic products. The final steps decisive for glucose release (fructose 1,6‐bisphosphatase and glucose 6‐phosphatase) appear to be highly dependent on posttranscriptional regulation according to actual glucose status. Glucogenic precursor entry, together with hepatic glycogen dynamics, is mostly sufficient to meet the needs for hepatic glucose output except in high‐producing dairy cows during the transition from the dry period to peak lactation. Lactating cows adapt to the increased glucose requirement for lactose production by mobilization of endogenous glucogenic substrates and increased hepatic PC expression. If these adaptations fail, lipid metabolism may be altered leading to fatty liver and ketosis. Increasing feed intake and provision of glucogenic precursors from the diet are important to ameliorate these disturbances. An improved understanding of the complex mechanisms underlying gluconeogenesis may further improve our options to enhance the postpartum health status of dairy cows.

Whey and casein labeled with l-[1-13C]leucine and muscle protein synthesis: effect of resistance exercise and protein ingestion

Muscle protein turnover following resistance exercise and amino acid availability are relatively well described. By contrast, the beneficial effects of different sources of intact proteins in relation to exercise need further investigation. Our objective was to compare muscle anabolic responses to a single bolus intake of whey or casein after performance of heavy resistance exercise. Young male individuals were randomly assigned to participate in two protein trials (n = 9) or one control trial (n = 8). Infusion of l-[1-(13)C]leucine was carried out, and either whey, casein (0.3 g/kg lean body mass), or a noncaloric control drink was ingested immediately after exercise. l-[1-(13)C]leucine-labeled whey and casein were used while muscle protein synthesis (MPS) was assessed. Blood and muscle tissue samples were collected to measure systemic hormone and amino acid concentrations, tracer enrichments, and myofibrillar protein synthesis. Western blots were used to investigate the Akt signaling pathway. Plasma insulin and branched-chain amino acid concentrations increased to a greater extent after ingestion of whey compared with casein. Myofibrillar protein synthesis was equally increased 1-6 h postexercise after whey and casein intake, both of which were higher compared with control (P < 0.05). Phosphorylation of Akt and p70(S6K) was increased after exercise and protein intake (P < 0.05), but no differences were observed between the types of protein except for total 4E-BP1, which was higher after whey intake than after casein intake (P < 0.05). In conclusion, whey and casein intake immediately after resistance exercise results in an overall equal MPS response despite temporal differences in insulin and amino acid concentrations and 4E-BP1.

Effects of microbial inoculants on corn silage fermentation, microbial contents, aerobic stability, and milk production under field conditions

The present study aimed to investigate the effects of 2 corn silage inoculation strategies (homofermentative vs. heterofermentative inoculation) under field conditions and to monitor responses in silage variables over the feeding season from January to August. Thirty-nine commercial dairy farms participated in the study. Farms were randomly assigned to 1 of 3 treatments: control (nonactive carrier; Chr. Hansen A/S, Hørsholm, Denmark), Lactisil (inoculation with 1 x 10(5)Lactobacillus pentosus and 2.5 x 10(4)Pediococcus pentosaceus per gram of fresh matter; Chr. Hansen A/S), and Lalsil Fresh (inoculation with 3 x 10(5)Lactobacillus buchneri NCIMB 40788 per gram of fresh matter; Lallemand Animal Nutrition, Blagnac, France). Inoculation with Lactisil had no effects on fermentation variables and aerobic stability. On the contrary, inoculation with Lalsil Fresh doubled the aerobic stability: 37, 38, and 80+/-8h for control, Lactisil, and Lalsil Fresh, respectively. The effect of Lalsil Fresh on aerobic stability tended to differ between sampling times, indicating a reduced difference between treatments in samples collected in April. Lalsil Fresh inoculation increased silage pH and contents of acetic acid, propionic acid, propanol, propyl acetate, 2-butanol, propylene glycol, ammonia, and free AA. The contents and ratios of DL-lactic acid, L-lactic acid relative to DL-lactic acid, free glucose, and DL-lactic acid relative to acetic acid decreased with Lalsil Fresh inoculation. Lalsil Fresh inoculation increased the silage counts of total lactic acid bacteria and reduced yeast counts. The Fusarium toxins deoxynivalenol, nivalenol, and zearalenone were detected in all silages at all collections, but the contents were not affected by ensiling time or by inoculation treatment. The effect of inoculation treatments on milk production was assessed by collecting test-day results from the involved farms and comparing the actual milk production with predicted milk production within farm based on test-day results from 2007 and 2008. The average milk production of lactating cows at test days during the study (January to September 2009) was 30.7+/-0.5 kg of energy-corrected milk/d. Milk production was 104.6+/-0.7% of the predicted yield and did not differ among treatments. In conclusion, the present study showed that homofermentative inoculants might not compete efficiently or might not deviate sufficiently from the epiphytic flora on whole-crop corn to affect fermentation in standard qualities of corn silage. Heterofermentative inoculation increased aerobic stability and numerous fermentation variables. None of the treatments affected milk production, and more-stable corn silage seemed to have a similar production value as compared with less-stable homofermented silage. Heterofermented silage can be evaluated for its properties to limit aerobic silage deterioration in the feed chain.

Portal Recovery of Intraruminally Infused Short-chain Fatty Acids in Sheep

Abstract Three rumen fistulated and catheterized sheep were used to study portal recovery of continuously intraruminally infused short-chain fatty acids (SCFA). Each sheep received six treatments: 0, 60, 120, 180, 240 and 300 mmol h−1 SCFA infused for 8 h in a semi-fasted state. The infused SCFA had a molar proportion of 65:20:15 (acetate:propionate:butyrate). [1 - 13C]Na-acetate was infused intravenously during the experiment for measurement of portal drained viscera (PDV) metabolism and irreversible loss rate (ILR) of acetate. Portal blood flow was measured by ultrasonic flowprobes. Portal blood, arterial blood and rumen liquid were sampled during the last 4 h of the experiments. A model of individual metabolite fluxes in the portal drained viscera is presented. The absorption rate of acetate, measured as portal net appearance plus PDV metabolism, and irreversible loss rate of acetate showed a linear response to infusion rate and accounted for 54 ± 8% and 73 ± 9% of infused acetate, respectively. The B-...

Quantification of whole blood short-chain fatty acids by gas chromatographic determination of plasma 2-chloroethyl derivatives and correction for dilution space in erythrocytes.

A precise, accurate and stable method for quantification of short-chain fatty acids (SCFA) in ovine whole blood is presented and validated. The method is based on esterification of plasma SCFA by reaction with chloroethyl chloroformate in a water/acetonitrile/2-chloroethanol solution and gas chromatographic (GC) analysis of the derivatives. Whole blood concentrations of SCFA could be obtained by correcting plasma concentrations for a 45% dilution space of SCFA in the erythrocyte fraction of the blood. The recovery of SCFA in plasma and whole blood was 96-100% independent of the haematocrit value when compared with water standards. The method avoided carry-over from sample to sample, contrary to earlier published methods. The average intra-assay and inter-assay coefficient of variation for repeated measurement of SCFA content in plasma samples was 2.5% and 3.1%, respectively. The derivative was found to be suitable for a precise and accurate determination of the 13

Feed-induced changes in the transport of butyrate, sodium and chloride ions across the isolated bovine rumen epithelium.

A short-chain fatty acid (SCFA) burst feeding strategy to dry cows (periodically high SCFA concentration in the ruminal fluid) has been hypothesized to increase the ruminal surface area and absorptive capacity. The present paper deals with the effects of a SCFA burst feeding strategy on epithelial transport function in dry cows. The epithelial transport capacity for [14C]butyrate and Na+ across the rumen epithelium in vitro was significantly increased by the SCFA burst feeding strategy, compared with the controls. The transport of Cl- was significantly increased by the SCFA burst feeding strategy at the high feeding level, but non-significantly decreased at the low feeding level. The increase in transport capacity could not be attributed to changes in epithelial resistance, to the concentration of Na+/K+-ATP-ase or to the epithelial surface area and structure (data on epithelial surface area and structure were published by Andersen et al., Report No. 23 from the National Institute of Animal Science, Denmark, 1994). The observed changes in transport must reflect a change in either composition of the cell membranes or processes that facilitate transport through the cell, including metabolic processes. The presented results support the existence of a saturable SCFA transport system in the epithelial cells and the results show that the capacity of this transport system can be the subject for feed-induced regulation.

Effect of composition of ruminally-infused short-chain fatty acids on net fluxes of nutrients across portal-drained viscera in underfed ewes.

Four ewes, each fitted with a rumen cannula and with catheters in the mesenteric artery and portal and mesenteric veins, received continuous intrarumen infusions of water or of short-chain fatty acids (SCFA). SCFA infusions were isoenergetic (83 kJ/h) and provided rumen molar proportions (acetate:propionate:butyrate) of 70:20:10, 50:40:10 or 50:20:30. The rumen SCFA production rate with the basal diet was 90.0, 23.1 and 8.8 mmol/h for acetate, propionate and butyrate respectively. Portal net fluxes indicated that 74, 67 and 22-30% of infused acetate, propionate and butyrate respectively, reached the portal vein. Portal net release of beta-hydroxybutyrate increased with SCFA infusions, irrespective of the amount of butyrate infused. Portal net release of lactate decreased with high-butyrate infusion. Portal net uptake of glucose increased with the SCFA infusions. In ewes infused with water, a portal net uptake of total amino acids (AA) was observed. SCFA infusions decreased the uptake of nonessential AA (glutamate, glycine, but not glutamine) and increased the net release of tyrosine and essential AA (isoleucine, leucine). Portal net fluxes of AA were similar with both high-acetate and high-propionate infusions. Lower net uptake of glutamine and net release of most essential AA and some nonessential AA were observed with the high-butyrate infusion. Energetic summation of portal net release was not significantly different between the three SCFA infusions, although it tended to be lower with high-butyrate infusion. This may be related to the higher trophic effect of butyrate on the digestive mucosa.

Effect of dietary nitrogen content and intravenous urea infusion on ruminal and portal-drained visceral extraction of arterial urea in lactating Holstein cows

Urea extraction across ruminal and portal-drained visceral (PDV) tissues were investigated using 9 rumen-cannulated and multi-catheterized lactating dairy cows adapted to low-N (12.9% crude protein) and high-N (17.1% crude protein) diets in a crossover design. The interaction between adaptation to dietary treatments and blood plasma concentrations of urea was studied by dividing samplings into a 2.5-h period without urea infusion followed by a 2.5-h period with primed continuous intravenous infusion of urea (0.493+/-0.012 mmol/kg of BW per h). Cows were sampled at 66+/-14 and 68+/-12 d in milk and produced 42+/-1 and 36+/-1 kg of milk/d with the high-N and low-N diets, respectively. The arterial blood urea concentration before urea infusion was 1.37 and 4.09+/-0.18 mmol/L with low-N and high-N, respectively. Dietary treatment did not affect the urea infusion-induced increase in arterial urea concentration (1.91+/-0.13 mmol/L). Arterial urea extraction across the PDV and rumen increased from 2.7 to 5.4+/-0.5% and from 7.1 to 23.8+/-2.1% when cows were changed from high-N to low-N, respectively. Urea infusion did not decrease urea extractions, implying that urea transport rates were proportional to arterial urea concentrations. Urea extraction increased more across the rumen wall than across the total PDV for low-N compared with high-N, which implies that a larger proportion of total PDV uptake of arterial urea is directed toward the rumen with decreasing N intake. The ruminal vein - arterial (RA) concentration difference for ammonia increased instantly (first sampling 15 min after initiation of infusion) to the primed intravenous infusion when cows were adapted to the low-N diet. The RA difference for ammonia correlated poorly to the ventral ruminal concentration of ammonia (r=0.55). Relating the RA difference for ammonia to a function of both ruminal ammonia concentration and the RA difference for urea markedly improved the fit (r=0.85), indicating that a large fraction of ammonia released to the ruminal vein is absorbed from an epithelial ammonia pool not in equilibrium with the ventral ruminal ammonia pool. Changing cows from high-N to low-N affected the relative blood urea clearance by kidneys and PDV. The clearance by the kidneys decreased from 41 to 27+/-2 L/h and the clearance by the PDV increased from 52 to 105+/-12 L/h when the diet was changed from high-N to low-N. In conclusion, urea transport across gut epithelia in cattle is adapting to N status and driven by mass action. Data are commensurable with a model for urea transport across gut epithelia based on regulated expression or activity of facilitative urea transporters.

Effect of abomasal glucose infusion on splanchnic and whole-body glucose metabolism in periparturient dairy cows

Six periparturient Holstein cows fitted with ruminal cannulas and permanent indwelling catheters in the hepatic portal vein, hepatic vein, mesenteric vein, and an artery were used to study the effects of abomasal glucose infusion on splanchnic and whole-body glucose metabolism. The experimental design was a split plot, with cow as the whole plot, treatment as the whole-plot factor, and days in milk (DIM) as the subplot factor. Cows were assigned to 1 of 2 treatments: the control (no infusion) or infusion (1,500 g/d of glucose infused into the abomasum from the day of calving). Cows were sampled at 12 d prepartum and at 4, 15, and 29 DIM. To study portal-drained visceral uptake of arterial glucose, [U-(13)C]glucose was continuously infused into the jugular vein on sampling days. Postpartum, voluntary dry matter intake and milk yield increased at a lower rate with the infusion compared with the control. The net portal flux of glucose increased with the infusion compared with the control, and 67 +/- 5% of the infused glucose was recovered as increased portal flux of glucose. The net hepatic flux of glucose was lower with the infusion compared with the control; however, the net hepatic flux of glucose per kilogram of dry matter intake was not affected by treatment. The arterial concentrations of glucose and insulin decreased and concentrations of nonesterified fatty acids increased from prepartum to 4 DIM with the control, but these effects were not observed with the infusion. The arterial concentration of beta-hydroxybutyrate decreased more from prepartum to 4 DIM with the infusion, compared with the control. Uptake of arterial [U-(13)C]glucose in the portal-drained viscera was affected neither by the infusion nor by the DIM and averaged 2.5 +/- 0.2%. The whole-body glucose supply changed to be less dependent on the recycling of lactate (Cori cycle) with the infusion. It was concluded that small intestinal glucose absorption is an efficient source of glucose to the peripheral tissues of dairy cows in very early lactation. At least 67% of the available glucose was recovered in the portal vein without affecting hepatic gluconeogenesis. Infused cows produced less milk and had a lower feed intake, indicating that an improved glucogenic status in very early lactation impaired metabolic adaptations to lactation.

Effects of ruminal ammonia and butyrate concentrations on reticuloruminal epithelial blood flow and volatile fatty acid absorption kinetics under washed reticulorumen conditions in lactating dairy cows

The effect of reticuloruminal epithelial blood flow on the absorption of propionate as a volatile fatty acid (VFA) marker in 8 lactating Holstein cows was studied under washed rumen conditions. The cows were surgically prepared with ruminal cannulas and permanent catheters in an artery and mesenteric, right ruminal, and hepatic portal veins. The experiment was designed with 2 groups of cows: 4 cows adapted to high crude protein (CP) and 4 to low CP. All cows were subjected to 3 buffers: butyric, ammonia, and control in a randomized replicated 3 × 3 incomplete Latin square design. The buffers (30 kg) were maintained in a temporarily emptied and washed rumen for 40 min. The initial concentration of VFA was 84.2 mmol/L. Butyrate was increased from 4 to 36 mmol/L in butyric buffer by replacement of acetate, and ammonia (NH(3)) was increased from 2.5 to 22.5 mmol/L in ammonia buffer by replacement of NaCl. Increasing amounts of deuterium oxide (D(2)O) were added to the buffers as the order of buffer sequence increased (6, 12, and 18 g of D(2)O). Ruminal clearance of D(2)O was used to estimate epithelial blood flow. To increase accuracy of the epithelial blood flow estimates, data of ruminal liquid marker (Cr-EDTA), and initial and final buffer volumes were fitted to a dynamic simulation model. The model was used to estimate ruminal liquid passages, residual liquid, and water influx (saliva and epithelia water) for each combination of cow and buffer (n=24). Epithelial blood flow increased 49±11% for butyric buffer compared with control. The ruminal disappearance of propionate (marker VFA) was affected by buffer and followed the same pattern as for epithelial blood flow. The correlation between ruminal disappearance of propionate and epithelial blood flow (r=0.56) indicates that the removal of propionate can be limited by epithelial blood flow. The ruminal disappearance of propionate increased 30±12% for the butyric compared with ammonia buffer and 12.5±8% when compared with control. The net portal flux of propionate increased 32±6% in butyric compared with control. In conclusion, rumen epithelial blood flow is positively correlated with ruminal disappearance of propionate and affects the kinetics of ruminal VFA absorption.