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The objectives of this study were to: (a) select an ideal organogel for the oil phase of a novel gel encapsulation technology, (b) optimize the formulation of an organogel and sodium alginate-based gel complex, and (c) examine the rumen protective ability of the gel by measuring 48-h in vitro ruminal dry matter disappearance and gas production from encapsulated dried and ground holy basil leaves.A rice-bran wax and canola oil organogel was selected for the oil phase of the gel complex as this combination had a 48-h dry matter disappearance of 6%, the lowest of all organogels analyzed. The gel complex was formulated by homogenizing the organogel with a sodium alginate solution to create a low-viscosity oil-in-water emulsion. Average dry matter disappearance of gel-encapsulated holy basil was 19%, compared to 42% for the free, unprotected holy basil. However, gel encapsulation of holy basil stimulated gas production. Specifically, gas production of encapsulated holy basil was four times higher than the treatment with holy basil added on top of the gel prior to incubation rather than encapsulated within the gel.Although the gel itself was highly degradable, it is speculated encapsulation thwarted holy basil's antimicrobial activity. © 2018 Society of Chemical Industry.

Dietary restriction (DR) increases median life span and protects against age-related disease. Improved longevity can be achieved by restriction of dietary energy, protein, or amino acids (AAs), such as methionine (Met). Met requirements have been defined using methodologies that measure the dose response to Met when all other dietary variables are held constant and with outcomes focused on protein turnover. Here, we cover protein and sulfur AA requirements and discuss the terms "deficient," "optimal," and "excess" and how these need to be considered. We additionally discuss the effect of methyl-donating compounds on sulfur AA metabolism and outcomes. We will discuss how the mechanistic target of rapamycin complex 1 (mTORC1) signaling network regulates protein turnover, lipogenesis and cell growth, proliferation, differentiation, and metabolism in response to hormones, AAs, and cellular energy status. Inhibition of mTORC1 signaling with rapamycin or genetic mutation increases median life span in model organisms, and mTORC1 inhibition may be responsible for some of the life span-extending effects of DR. Finally, we discuss how the sulfur AAs may regulate aspects of reactive oxygen species (ROS) mitigation. Overall, we suggest that approaches evaluating AA intake need to consider whole-body protein synthesis and measures related to tissue-specific and whole-body metabolism that have been associated with longevity.

Background: Including ammonia in low-crude protein (CP) diets deficient in dispensable amino acid (DAAs) increases nitrogen retention in growing pigs.Objective: We investigated the absorption and metabolism of dietary ammonia nitrogen in the portal-drained viscera (PDV) and liver of pigs fed a diet deficient in DAA nitrogen.Methods: Eight pigs with an initial mean ± SD body weight (BW) of 26.5 ± 1.4 kg were surgically fitted with 4 catheters each (portal, hepatic and mesenteric veins, and carotid artery). The pigs were fed (2.8 × 191 kcal/kg BW0.60), for 7 d and every 8 h, a diet deficient in DAA nitrogen supplemented with increasing amounts of ammonia nitrogen (CP: 7.76%, 9.27%, and 10.77%; indispensable amino acid nitrogen:total nitrogen ratio: 0.71, 0.59, and 0.50 for control and low- and high-ammonia diets, respectively). The treatment sequence was based on a Latin square design with 3 consecutive periods. On the last day of each period, blood flows in the portal and hepatic veins were determined with a continuous infusion of ρ-amino hippuric acid into the mesenteric vein. Serial blood samples were taken to determine ammonia and urea nitrogen concentration. Net balances of ammonia and urea nitrogen were calculated for the PDV and liver.Results: Cumulative (8 h) ammonia nitrogen appearance in the portal vein increased (P ≤ 0.05) with ammonia intake (433, 958, and 1629 ± 60 mg ammonia nitrogen/meal for control and low- and high-ammonia diets, respectively). The cumulative hepatic uptake of ammonia nitrogen increased (P ≤ 0.05) with ammonia nitrogen supply. The cumulative urea nitrogen appearance in the hepatic vein tended to increase (P ≤ 0.10) only in high-ammonia treatment (-92.5, -59.4, and 209.7 ± 92 mg urea nitrogen/meal for control and low- and high-ammonia diets, respectively) and, relative to the control diet, represented -6.0% and 11% of ammonia nitrogen intake.Conclusion: Dietary ammonia nitrogen is poorly utilized for urea production across splanchnic organs when pigs are fed diets deficient in DAA nitrogen.

Background Consumption of dairy products reduces risk of type 2 diabetes. Milk proteins and fats exhibit anti-diabetic properties but milk sugars have been studied little in this context. Galactose from milk lactose is readily converted to glycogen in the liver but its effects on insulin sensitivity have not been assessed. Prebiotic oligosaccharides from milk alter gut microbiota and can thereby influence host metabolism. Our objective was to assess the effect on insulin sensitivity of dietary galactose compared to glucose and fructose, and fermentable galacto-oligosaccharides compared to non-fermentable methylcellulose. Methods Diets containing 15% of dry matter from glucose, fructose, galactose, galacto-oligosaccharides, or methylcellulose were fed to 36 rats per diet for 9 weeks. Hyperinsulinemic-euglycemic clamps with [3-3H]glucose infusion and a steady-state 2-[1-14C]deoxyglucose bolus injection were used to assess insulin sensitivity and glucose uptake indices. Tissue was collected in fed, fasted and fasted, insulin-stimulated states. Results Galactose increased glucose infusion rate during the clamp by 53% and decreased endogenous glucose production by 57% compared to glucose and fructose. Fed-state hepatic glycogen content was greater with galactose compared to glucose and fructose, consistent with a potentiation of the insulin effect on glycogen synthase by dephosphorylation. Galactose decreased the fecal Firmicutes:Bacteroidetes ratio while galacto-oligosaccharides increased abundance of fecal Bifidobacterium spp. 481-fold compared to methylcellulose, and also increased abundance of Lactobacillus spp. and Bacteroidetes. Galacto-oligosaccharides did not affect glucose infusion rate or endogenous glucose production during basal or clamp periods compared to methylcellulose. Conclusions Galactose at 15% of daily intake improved hepatic insulin sensitivity in rats compared to glucose and fructose. Galactose caused an increase in fed-state hepatic glycogen content and a favourable shift in gut microbial populations. Intake of galacto-oligosaccharides improved the gut microbial profile but did not improve insulin sensitivity.

The phosphorylation of mammalian target of rapamycin complex 1 (mTORC1) components and integrated stress response networks in the mammary glands of lactating cows have not accounted for the stimulation of milk protein yield by chronic supplementation with AA or glucose. Faster milk protein synthesis could be a consequence of increased milk protein mRNA per cell, the number of ribosomes per cell, the secretory capacity of cells, or the mammary cell number. To investigate these 4 possibilities using a translational and transcriptional approach, we performed protein and gene expression analyses of mammary and longissimus dorsi tissue collected from lactating dairy cows after 5 d of abomasal infusion with saline or 844 or 1,126 g/d of an essential AA (EAA) mixture, with and without 1,000 g/d glucose. Infusion with EAA increased milk protein yield but did not affect the phosphorylation of mTORC1-related proteins in the mammary gland. In skeletal muscle, phosphorylation of 4EBP1 (eIF4E-binding protein 1) increased in response to both EAA and glucose, and phosphorylated S6K1 (70-kDa ribosomal protein S6 kinase) increased with glucose. In response to EAA, mammary mRNA expression of the marker genes for milk proteins, ribosome biogenesis, and cell proliferation were not upregulated. Instead, reciprocal regulation of 2 arms of the unfolded protein response occurred. Infusion of EAA for 5 d activated XBP1 (X-box binding protein 1) mRNA, encoding a transcription factor for endoplasmic reticulum biogenesis, and it decreased the mRNA expression of genes encoding pro-apoptotic protein CHOP (C/EBP homologous protein) and downstream GADD34 (growth arrest and DNA damage-inducible 34). These findings implicate non-stress-related, adaptive capabilities of the unfolded protein response in the long-term nutritional regulation of milk protein yield in lactating dairy cows.

Two studies were conducted using adult dogs to evaluate the effect of increasing the inclusion of soybean meal (SBM) in an adult dog food on body com- position, hematological and biochemical blood analyses, and total tract nutrient digestibility. Nutritionally complete and balanced diets were formulated with commercial- grade SBM (48% CP) to replace 0, 10, 20, or 30% of the protein provided by dried chicken protein resulting in final SBM inclusion of 0, 6.0, 11.5, and 17.0% (as-fed basis), respectively. In study 1, diets were fed during a 24-wk feeding trial using 36 female (spayed), adult hounds to evaluate food intake, BW, body composition, and blood measurements. There were no diet-related differences in food intake or BW. Body composition responded in a quadratic manner to increased dietary SBM inclusion with the percentage (%) of lean mass responding posi- tively (P < 0.05) and absolute amounts of fat mass and percent body fat responding negatively (P < 0.05). All diagnostic blood components remained within normal physiological ranges for healthy, adult dogs. Serum con- centrations of C-reactive protein and IGF-1 were similar among diets. In study 2, diets were evaluated in a digest- ibility study using 12 adult dogs in a 4 × 4 Latin square design. Increased SBM inclusion was associated with lin- ear increases in the digestibility of CP (P < 0.05) and fat (P < 0.05) and CP retention (P < 0.05). Linear reductions in fecal DM content (P < 0.01) and increased fecal output (P < 0.05) were noted with increased SBM inclusion. All diets were similar in DE and ME content, but a quadratic trend was noted with increased SBM inclusion when DE (P = 0.083) and ME (P = 0.062) were expressed per unit of metabolic body size. Overall, it can be inferred from these results that the partial replacement of dried chicken protein with SBM in a nutritionally complete and bal- anced diet does not compromise the nutritional status and long-term health of adult dogs.

To test which, if any, of the major milk precursors can elicit a rapid change in the rate of mammary blood flow (MBF) and to define the time course and magnitude of such changes, 4 lactating cows were infused with glucose, amino acids, or triacylglycerol into the external iliac artery feeding one udder half while iliac plasma flow (IPF) was monitored continuously by dye dilution. Adenosine and saline were infused as positive and negative controls, respectively, and insulin was infused to characterize the response to a centrally produced anabolic hormone. To test the roles of cyclooxygenase, NO synthase and ATP-sensitive K (KATP) channels in nutrient-mediated changes in blood flow, their respective inhibitors-indomethacin, Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME), and glibenclamide-were infused simultaneously with glucose. Each day, 1 infusate was given twice to each cow, over a 20-min period each time, separated by a 20-min washout period. In addition, each treatment protocol was administered on 2 separate days. A 73% increase in IPF during adenosine infusion showed that the mammary vasodilatory response was quadratic in time, with most changes occurring in the first 5min. Glucose infusion decreased IPF by 9% in a quadratic manner, most rapidly in the first 5min, indicating that a feedback mechanism of local blood flow control, likely through adenosine release, was operative in the mammary vasculature. Amino acid infusion increased IPF 9% in a linear manner, suggesting that mammary ATP utilization was stimulated more than ATP production. This could reflect a stimulation of protein synthesis. Triacylglycerol only tended to decrease IPF and insulin did not affect IPF. A lack of IPF response to glibenclamide indicates that KATP channels are not involved in MBF regulation. Indomethacin and L-NAME both depressed IPF. In the presence of indomethacin, glucose infusion caused a quadratic 9% increase in IPF. Indomethacin is an inhibitor of mitochondrial function, so the glucose-induced increase in IPF was interpreted as feedback on mammary adenosine release from an anabolic response to glucose. Because NO synthase was not inhibited during indomethacin infusion, the feedback system is postulated to act through endothelial NO synthase. In the presence of L-NAME, glucose infusion had no effect on IPF, indicating that endothelial cyclooxygenase is not involved in glucose-induced changes in MBF.

One hundred and two cross-bred steer calves (BW=368±6.6 kg) were used in a 2×3 factorial arrangement of treatments plus a control to determine the effect of inclusion level and form of corn distillers grains plus solubles (DGS) on finishing performance, carcass characteristics, and feeding behavior using whole corn grain-based finishing diets. The DGS were fed at 0 (control), 167, 333, and 500 g/kg of diet DM using dry (DDGS) or modified wet (MWDGS) DGS. All diets contained 100 g/kg haylage as a forage source, and were formulated to contain at least 137 g/kg CP. Individual intake and feeding behavior was measured using the Insentec system. Cattle were fed until ultrasound backfat thickness reached 10 mm. Data were analyzed using GLM of SAS; treatment means were compared using contrast statements (control vs. others, DDGS vs. MWDGS, inclusion level of DGS (linear, quadratic), and interactions between form and linear and quadratic inclusion level). There were no effects (P>0.05) of dietary treatment on final BW, ADG, days on feed, dressing yield (g hot carcass weight/kg final BW), hot carcass weight, marbling score, lean yield, and lean color. Rumen pH at slaughter linearly increased (P=0.001) and liver abscess score linearly decreased (P=0.03) with increasing DGS inclusion. There were significant quadratic form×level interactions (P≤0.03) for time at feeder, time per visit, number of meals, meal size, and eating rate. Number of meals (meals/d) linearly increased (P=0.006) with increasing DGS inclusion and was greater (P=0.006) in cattle fed MWDGS than those fed DDGS. Meal size linearly decreased (P=0.002) with increasing DGS inclusion and was greater (P=0.002) in cattle fed DDGS than those fed MWDGS. Finishing performance and carcass traits were generally not affected by feeding DDGS or MWDGS up to 500 g/kg diet DM in whole corn grain-based finishing diets. However, liver abscess scores were reduced with DGS inclusion and feeding behavior was influenced by form and level of DGS.

In the present study, fifteen growing pigs were used to determine the whole-body oxidation, retention efficiency (RE) and apparent conversion (AC) of α-linolenic acid (18 : 3n-3) ton-3 highly unsaturated fatty acids (HUFA), including EPA (20 : 5n-3) and DHA (22 : 6n-3). The pigs were fed a diet containing 10 % flaxseed for 30 d. Whole-body fatty acid composition was determined at initial (27·7 (se1·9) kg), intermediate (day 15; 39·2 (se1·4) kg) and final (45·7 (se2·2) kg) body weight. On day 12, four pigs were fed 10 mg/kg of uniformly labelled13C-18 : 3n-3 (single-bolus dose) to determine the oxidation of 18 : 3n-3. Expired$$CO_{2} $$samples were collected for 24 h thereafter. The whole-body content ofn-3 PUFA increased linearly (Pn-3 exhibited a quadratic response (Pn-3 tended to reduce with time (P= 0·098). The AC of ingested 18 : 3n-3 to the sum ofn-3 HUFA was reduced with time (Pv.7·53 % for days 0–15 and days 15–30, respectively). The AC of 18 : 3n-3 to 20 : 5n-3 or 22 : 6n-3 was lower than that to 20 : 3n-3, both for days 0–15 (Pv.7·06 %) and for days 15–30 (Pv.4·29 %). The direct oxidation of 18 : 3n-3 was 7·91 (se0·98) % and was similar to the calculated disappearance of 18 : 3n-3 between days 0 and 30 (8·81 (se5·24) %). The oxidation of 18 : 3n-3 was much lower than that reported in other species. The AC of 18 : 3n-3 ton-3 HUFA was reduced over time and that to 20 : 3n-3 in the present study was much higher than that reported in other species and should be explored further.

Removing His from a postruminal AA infusion decreases milk protein and increases milk fat content. Feather meal is an inexpensive protein source, high in rumen undegradable protein but low in His. The objective of our study was to investigate dietary feather meal as a method for creating a His deficiency or imbalance to alter milk composition. Four dietary treatments were fed for 4 wk each to 8 multiparous mid-lactation Holstein cows in a replicated 4 × 4 Latin square design. A standard-protein control diet (SP-C) was formulated to provide 3,100g/d of metabolizable protein (MP). Feather meal was added to the control diet either to replace the MP isonitrogenously (SP-FM) or to increase the MP supply to 3,484 g/d (HP-FM). As an isonitrogenous control for HP-FM, a high-protein diet (HP-C) was formulated with His-adequate protein sources to provide the same MP content as HP-FM. Dry matter intake tended to decrease when feather meal was fed. Predicted flows of digestible His, Met, and Lys, and plasma concentrations of these AA were reduced on both feather meal diets. Predicted flows of total digestible essential AA were not different between HP-FM and SP-C. We concluded that the DMI depression on HP-FM prevented an imbalance of excess AA over His, and created a deficiency of His, Met, and Lys compared with SP-C. Milk production decreased on the 2 feather meal treatments, partly explained by a tendency for DMI to decrease. Milk yield was lowest on SP-FM at 30.3 kg/d and highest on HP-C at 37.9 kg/d. Milk fat yield was not affected by diet but protein and lactose yields were both lower with feather meal. Protein yields were 860 and 998 g/d, whereas lactose yields were 1,384 and 1,561 g/d for SP-FM and HP-FM, respectively. This resulted in a higher fat content and lower protein percentage on FM diets. The ratio of solids-not-fat:fat in milk was lowest on SP-FM at 2.11 compared with 2.56 on SP-C. Adding feather meal to the diet by replacing MP isonitrogenously was more effective at lowering the solids-not-fat:fat ratio than increasing the MP content with an imbalanced protein source.

The characterization of blood metabolite concentrations over the circadian period and across physiological stages is important for understanding the biological basis of feed efficiency, and may culminate in indirect methods for assessing feed efficiency. Hematological analyses for albumin, urea, creatine kinase, glutamate dehydrogenase, aspartate aminotransferase, carbon dioxide, and acetate were carried out in growing and gestating heifers. These measures were carried out in a sample of 36 Bos taurus crossed beef heifers held under the same husbandry conditions. Hourly blood samples were collected over a 24-h period on three separate sampling occasions, corresponding approximately to the yearling (and open), early-gestation and late-gestation stages. This design was used to determine variation throughout the day, effects due to physiological status and any associations with feed efficiency, as measured by residual feed intake. Blood analyte levels varied with time of day, with the most variation occurring between 0800 and 1600 h. There were also considerable differences in analyte levels across the three physiological stages; for example, creatine kinase was higher (P

The objective of this study was to determine if the addition of glycerol to the diet of dairy cows would stimulate milk protein yield in the same manner as the addition of corn grain. Twelve multiparous lactating dairy cows at 81 ± 5 d in milk were subjected to 3 dietary treatments in a replicated 3 × 3 Latin square design for 28-d periods. The diets were a 70% forage diet considered the basal diet, the basal diet with 19% ground and high-moisture corn replacing forages, and the basal diet with 15% refined glycerol and 4% added protein supplements to be isocaloric and isonitrogenous with the corn diet. Cows were milked twice a day and samples were collected on the last 7 d of each period for compositional analysis. Within each period, blood samples were collected on d 26 and 27, and mammary tissue was collected by biopsy on d 28 for Western blot analysis. Dry matter intake increased from 23.7 kg/d on the basal diet to 25.8 kg/d on the corn diet and 27.2 kg/d on the glycerol diet. Dry matter intake tended to be higher with glycerol than corn. Milk production increased from 39.2 kg/d on the basal diet to 43.8 kg/d on the corn diet and 44.2 kg/d on the glycerol diet. However, milk yield did not differ between corn and glycerol diets. Milk lactose yields were higher on the corn and glycerol diets than the basal diet. Milk fat yield significantly decreased on the glycerol diet compared with the basal diet and tended to decrease in comparison with the corn diet. Mean milk fat globule size was reduced by glycerol feeding. Milk protein yield increased 197 g/d with addition of corn to the basal diet and 263 g/d with addition of glycerol, and the glycerol effect was larger than the corn effect. The dietary treatments had no effects on plasma glucose concentration, but plasma acetate levels decreased 27% on the glycerol diet. Amino acid concentrations were not affected by dietary treatments, except for branched-chain amino acids, which decreased 22% on the glycerol diet compared with the corn diet. The decreases in plasma acetate and branched-chain amino acid concentrations with glycerol and the larger effects of glycerol than corn on milk protein and fat yields suggest that glycerol is more glucogenic for cows than corn grain.

Selenium (Se)-enriched milk provides antioxidant benefits and has therapeutic potential against cancer. However, both antidiabetic and prodiabetic effects have been attributed to Se. Our objective was to evaluate the effect of Se-enriched milk casein on insulin sensitivity in rats when given at the requirement of 0.25 ppm Se and supranutritionally on both low- and high-fat diets. Two hundred sixteen male Sprague-Dawley rats were fed low- or high-fat diets containing one, two or eight times the Se requirement in a randomized block design. After 7 weeks, 72 rats were subjected to the hyperinsulinemic-euglycemic clamp with [3-3H]glucose infusion to estimate glucose fluxes. Tissues were collected from the remaining 144 rats 8 min after ip saline or insulin injection. During hyperinsulinemic-euglycemic clamps, glucose infusion rate was 22% lower (P=.058), and endogenous glucose production was 76% higher (P=.054) when Se content increased from one to eight times the requirement on low-fat diets, indicating impaired hepatic insulin sensitivity. Se also decreased the ability for insulin to stimulate Akt phosphorylation at Thr308. Hepatic oxidation state and expression of selenoprotein P and glutathione peroxidase-1 were unaffected while expression of insulin receptor substrate (IRS)-1 and-2 and PPARγ coactivator-1α (PGC-1α) decreased with supranutritional Se and high-fat intake. In addition, hepatic expression of regulatory and catalytic subunits of phosphatidylinositol 3-kinase (PI3K) decreased with supranutritional intake of Se. Se intake from enriched casein up to eight times the requirement impairs hepatic insulin sensitivity in a mechanism similar to fat feeding, via attenuated IRS/PI3K/Akt signaling and decreased PGC-1α expression.

To support decision-making around diet selection choices to manage glycemia following a meal, a novel mechanistic model of intermittent gastric emptying and plasma glucose-insulin dynamics was developed. Model development was guided by postprandial timecourses of plasma glucose, insulin and the gastric emptying marker acetaminophen in infant calves fed meals of 2 or 4 L milk replacer. Assigning a fast, slow or zero first-order gastric emptying rate to each interval between plasma samples fit acetaminophen curves with prediction errors equal to 9% of the mean observed acetaminophen concentration. Those gastric emptying parameters were applied to glucose appearance in conjunction with minimal models of glucose disposal and insulin dynamics to describe postprandial glycemia and insulinemia. The final model contains 20 parameters, 8 of which can be obtained by direct measurement and 12 by fitting to observations. The minimal model of intestinal glucose delivery contains 2 gastric emptying parameters and a third parameter describing the time lag between emptying and appearance of glucose in plasma. Sensitivity analysis of the aggregate model revealed that gastric emptying rate influences area under the plasma insulin curve but has little effect on area under the plasma glucose curve. This result indicates that pancreatic responsiveness is influenced by gastric emptying rate as a consequence of the quasi-exponential relationship between plasma glucose concentration and pancreatic insulin release. The fitted aggregate model was able to reproduce the multiple postprandial rises and falls in plasma glucose concentration observed in calves consuming a normal-sized meal containing milk components.

Four crossbred steers (average BW = 478 ± 33 kg) were used in a 4 × 4 Latin square design to determine the effects of dietary concentration of dry corn distillers grains plus solubles (DDGS) in whole corn-based finishing diets on total tract digestion and nutrient balance and excretion. The DDGS were fed at 0% (control), 16.7%, 33.3%, and 50% of dietary DM. All diets contained 10% (DM basis) alfalfa/grass haylage and were formulated to meet or exceed the estimated requirements for CP. Steers were fed the experimental diets ad libitum for a 14-d adaptation period followed by a 5-d period for fecal and urine collection. Increasing concentration of DDGS in diets from 0 to 50% of DM linearly decreased (P 0.38) among treatments. Retention (g/d; intake minus urinary and fecal excretion) of N did not differ (P > 0.16) among treatments. Retention of P tended (P = 0.07) to linearly increase and retention of S (g/d) linearly increased (P = 0.004), with increasing DDGS concentration. There were no effects (P > 0.16) of dietary treatment on digestion and retention of Se, Mg, K, and Na. Plasma P and S concentrations increased (P = 0.03 and 0.01, respectively) with increasing DDGS concentration. These data indicate that feeding DDGS up to 50% of dietary DM in whole corn grain-based finishing diets does not have a negative effect on nutrient retention but decreases digestibility. Total excretion of N, P, Ca, Mg, S, and K increased as DDGS concentration increased.

Summary There are few reported estimates of amino acid (AA) kinetics in adult mammals and none exist in adult dogs. The study objectives were to evaluate the use of oral isotope delivery in contrast to the more commonly used intravenous (IV) delivery to estimate AA kinetics in adult dogs and to estimate splanchnic extraction and gastric emptying using a commonly accepted mathematical model. Dogs received 25 × 1/2-hourly meals (13 g/kg BW/day) and either an oral or IV bolus of l-[1-13C]Phe (12 mg/kg BW). Blood samples were taken immediately before each feeding. Concentrations of plasma Phe were measured using liquid chromatography-tandem mass spectrometry. There were no differences in baseline plasma Phe concentrations (34 μm ± 0.61), Phe distribution volume, Phe pool size and rate constants between dogs when the tracer was administered IV or orally (p > 0.25). Decay curve for plasma l-[1-13C]Phe differed between IV and oral dosing protocols with IV dosing fit best using a two-compartment model. Phe disappeared from plasma at a mean rate of 2.8%/min. Estimates of gastric emptying and splanchnic extraction did not differ based on oral or IV tracer dosing when the decay curves were fit with the two-compartment model (p > 0.40). The half-life for gastric emptying was 18 min, and first-pass Phe extraction by the splanchnic bed was 24% of the dietary Phe. These results suggest that oral isotope dosing can be used as an alternative to IV isotope dosing in studies that utilize a primed, constant dosing approach to measure protein and amino acid kinetics.

The decline in mammary epithelial cell number as lactation progresses may be due, in part, to oxidative stress. Selenium is an integral component of several antioxidant enzymes. The present study was conducted to examine the effect of oxidative stress and selenomethionine (SeMet) on morphology, viability, apoptosis, and proliferation of bovine mammary epithelial cells (BMEC) in primary culture. Cells were isolated from mammary glands of lactating dairy cows and grown for 3 d in a low-serum gel system containing lactogenic hormones and 0 or 100 μM H2O2 with 0, 10, 20, or 50 nM SeMet. Hydrogen peroxide stress increased intracellular H2O2 to 3 times control concentrations and induced a loss of cuboidal morphology, cell-cell contact, and viability of BMEC by 25%. Apoptotic cell number more than doubled during oxidative stress, but proliferating cell number was not affected. Supplementation with SeMet increased glutathione peroxidase activity 2-fold and restored intracellular H2O2 to control levels with a concomitant return of morphology and viability to normal. Apoptotic BMEC number was decreased 76% below control levels by SeMet and proliferating cell number was increased 4.2-fold. These findings suggest that SeMet modulated apoptosis and proliferation independently of a selenoprotein-mediated reduction of H2O2. In conclusion, SeMet supplementation protects BMEC from H2O2-induced apoptosis and increased proliferation and cell viability under conditions of oxidative stress.

Milk protein yield (MPY) responses to dietary carbohydrates, particularly glucose, are partially regulated by protein synthetic pathways such as mechanistic target of rapamycin (mTORC1) and the integrated stress response (ISR) network. However, changes in these pathways have not been observed in response to prolonged glucose supplementation, despite observed changes in MPY. To determine if gene expression could identify alternative mechanisms of the MPY response to glucose, mammary and muscle biopsies were analyzed from two separate trials in which dairy cattle received supplemental glucose. In experiment 1, cows (n = 12) received 0 or 1 kg/d glucose I.V. in a switchback design. In experiment 2, cows (n = 5) received 0, 1.1, or 2.2 kg/d of glucose via abomasal infusion in a Latin square design. In both trials, glucose infusion did not affect DMI but increased milk and lactose yields. Glucose infusion significantly increased MPY 63 g/d in experiment one, and tended (P = 0.15) to increase MPY 82 g/d in experiment two. In mammary tissue, expression of genes related to protein translation, cell turnover, and cell differentiation were unaffected by infusion treatments (P > 0.15). Longissimus dorsi tissue was analyzed to determine if changes in muscle metabolism could support or interfere with glucose effects on milk protein synthesis in the mammary glands. Despite evidence of an insulin/glucose response in expression of genes related to glucose metabolism, no significant changes in genes related to protein turnover or amino acid metabolism were detected in skeletal muscle (P > 0.15). Overall, our findings suggest that glucose-induced changes in milk and component yields are not under transcriptional control of secretory cell activity or turnover, nor are there significant changes in muscle gene expression that support changes in amino acid supply for milk protein synthesis in the mammary glands.

Fermentative catabolism of dietary and endogenous amino acids (AA) in the upper gut of pigs (FAAC) can result in significant loss of AA available for protein synthesis and body maintenance functions. A continuous infusion trial was performed using isotope tracers to determine ammonia flux in the upper gut, whole body urea flux, urea recycling (urea flux − urinary urea excretion), and FAAC (ammonia flux in the upper gut − urea recycling) in ileal-cannulated growing pigs fed a control diet (C, 19.3% CP), the control diet with added fibre (F, 12% pectin added at expense of cornstarch), or a diet low in protein (LP, 13.6% CP). 15 N-ammonium chloride and 13 C-urea were infused intragastrically and intravenously, respectively, for a period of 4 days. In samples obtained on days 3 and 4 of infusion, 15 N-enrichments in blood urea (6.21 ± 1.65, 8.93 ± 2.01, and 9.78 ± 1.40 atoms percent excess (APE) for C, F and LP, respectively) were higher than those in ileal ammonia (0.44 ± 0.12, 0.37 ± 0.14, and 0.71 ± 0.10 APE). This suggests a rapid absorption of ammonia prior to the distal ileum and lack of uniformity for enrichment in the digesta ammonia pool. Simple isotope dilution calculations are, therefore, inadequate for calculating FAAC and ammonia flux in the upper gut of pigs. A two compartment (ileal ammonia and plasma urea) model was developed to determine possible value ranges for FAAC in the upper gut (0.0 to 13.3 ± 1.42, 15.5 ± 1.65, and 10.7 ± 1.20 mmol N/kg/d for the three treatments), but this model also has limitations. Quantifying FAAC in the upper gut of pigs offers a number of challenges, but warrants further investigation.

Initiation of mRNA translation and elongation of the polypeptide chain are 2 regulated processes responsible for the short-term postprandial acceleration of protein synthesis in animal tissues. It is known that a chronic increase in the absorptive supply of AA stimulates protein synthesis in ruminant animals, but effects on translation initiation and elongation are unknown. To determine whether initiation or elongation phases of global mRNA translation are affected by chronic elevation of AA supply, 24 ewe lambs of 25.9 +/- 2.5 kg of BW were randomly allocated to 4 treatment groups of 6 lambs each. All lambs received a basal diet of barley and hay at 1.2 times maintenance ME intake. Treatments were an intravenous (i.v.) saline infusion as a control, i.v. infusion of 6 essential AA (EAA; Arg, Lys, His, Thr, Met, Cys) for 10 d, i.v. infusion of the same EAA excluding Met and Cys (EAA-SAA) for 10 d, and an oral drench of fishmeal twice daily for 17 d. Fishmeal supplementation supplied an extra 719 mg of N x kg(-0.75) x d(-1) and N retention was increased 519 mg x kg(-0.75) x d(-1) over the control. The EAA treatment supplied an extra 343 mg of N x kg(-0.75) x d(-1) directly into the blood, and N balance was increased by 268 mg x kg(-0.75) x d(-1). Deletion of Met plus Cys from EAA had no effect on N balance. The results indicate that Met plus Cys did not limit body protein gain on the basal diet alone or the basal diet plus 6 AA. Protein fractional synthesis rates in liver, duodenum, skin, rumen, semimembranosus, and LM were measured by a flooding dose procedure using L-[ring-2,6-(3)H]-Phe. Ribosome transit times were estimated from the ratio of nascent to total protein-bound radioactivities. Fishmeal and EAA treatments had no effect on RNA, DNA, or protein contents of tissues, but fractional synthesis rate, translational efficiency, and concentrations of active ribosomes were consistently elevated. Ribosome transit time was not affected by long-term AA supply. We conclude that the chronic stimulation of protein synthesis by long-term i.v. infusion of EAA or supplementation with an undegradable protein source is brought about by an improvement in the rate of initiation of mRNA translation with no change in the rate of polypeptide chain elongation.

Production of protein by the lactating mammary gland is stimulated by intake of dietary energy and protein. Mass-action effects of essential amino acids (EAA) cannot explain all of the nutritional response. Protein synthesis in tissues of growing animals is regulated by nutrients through the mammalian target of rapamycin (mTOR) and integrated stress response (ISR) networks. To explore if nutrients signal through the mTOR and ISR networks in the mammary gland in vivo, lactating cows were feed-deprived for 22 h and then infused i.v. for 9 h with EAA+ glucose (Glc), Glc only, l-Met+l-Lys, l-His, or l-Leu. Milk protein yield was increased 33 and 27% by EAA+Glc and Glc infusions, respectively. Infusions of Met+Lys and His generated 35 and 41%, respectively, of the EAA+Glc response. Infusion of EAA+Glc reduced phosphorylation of the ISR target, eukaryotic initiation factor(eIF) 2, in mammary tissue and increased phosphorylation of the mTOR targets, ribosomal S6 kinase 1 (S6K1) and S6. Both responses are stimulatory to protein synthesis. Glucose did not significantly increase mammary S6K1 phosphorylation but reduced eIF2 phosphorylation by 62%, which implicates the ISR network in the stimulation of milk protein yield. In contrast, the EAA infusions increased (P < 0.05) or tended to increase (P < 0.1) mammary mTOR activity and only His, like Glc, decreased eIF2 phosphorylation by 62%. Despite activation of these protein synthesis signals to between 83 and 127% of the EAA+Glc response, EAA infusions produced less than one-half of the milk protein yield response generated by EAA+Glc, indicating that ISR and mTOR networks exert only a portion of the control over protein yield.

The nutritional and endocrine factors affecting protein translation in the bovine mammary gland, and the molecular mechanisms mediating their effects, are not well understood. The objective of this study was to assess the role of the mammalian target of rapamycin (mTOR) signaling pathway in the regulation of mammary protein synthesis by nutrients and hormones. Mammary epithelial acini isolated from lactating dairy cows were treated in medium containing, alone or in combination, a mixture of AA or glucose and acetate (GA) as energy substrates, or a combination of the lactogenic hormones hydrocortisone, insulin, and prolactin (HIP). Changes in the rate of mammary protein synthesis and the phosphorylation state of components of the mTOR signaling pathway were measured. Mammary protein synthesis was 50% higher with increased availability of AA in medium. The presence of GA or treatment of mammary acini with HIP alone did not affect mammary protein synthesis. The stimulation of mammary protein synthesis by AA was enhanced by HIP treatment, but not by the presence of GA in medium. Treatment of mammary acini with HIP induced the phosphorylation of protein kinase B. This effect was augmented in the presence of either AA or GA in medium. The stimulation of mammary protein synthesis by AA and its enhancement by HIP were associated with increased phosphorylation of the mTOR substrates, p70 ribosomal protein S6 kinase-1, and eukaryotic initiation factor 4E (eIF4E)-binding protein-1 (4E-BP1), and dissociation of 4E-BP1 from eIF4E. The results suggest that nutrients and hormones may modulate mammary protein synthesis through the mTOR signaling pathway.

Conjugated linoleic acid (CLA) has the potential to be used as a dietary means of manipulating milk fat production of dairy cows to meet specified short-term targets, as in a supply-managed industry. An experiment was conducted to evaluate the dose-response to calcium salts of CLA fed as a top-dress on a corn- and alfalfa-based TMR. In two 5 × 5 Latin squares of 28-d periods, five primiparous and five multiparous cows were fed 0, 22, 45, 67 or 178 g d-1 CLA (29% trans-10, cis-12) as a calcium salt. Results from one multiparous cow on the 178 g d-1 treatment were removed from statistical analysis because milk production fell to 1.2 kg d-1 by the end of the period. On average, milk, lactose and protein yields increased at the lower doses of CLA and decreased to control levels at the highest dose. Milk fat yield declined progressively from 876 to 770, 689, 676 and 543 g d-1 on the five doses. Net energy balance of cows increased linearly from -1.5 Mcal d-1 on the control to 2.0 Mcal d-1 at 178 g d-1 CLA. The percentage depression in milk fat yield at different doses of trans-10, cis-12 CLA was described by the equation y = 54/(1 + 22.8/dose). Comparison with published literature suggested that feeding calcium salts of CLA was 30% as effective in depressing milk fat production as an abomasal infusion, due to rumen biohydrogenation and intestinal indigestibility.Key words: Milk composition, cattle, diet

The current study investigates regulation of mRNA expression of components of ubiquitin-mediated proteolysis in transition dairy cows. Longissimus dorsi muscle (exp. 1) and liver (exp. 2) biopsies were collected from Holstein dairy cows at 27 and 16 d pre-partum, respectively, and 3 and 10 d post-partum, respectively. Regulation of C8, E2, and ubiquitin mRNA expression was determined. Upregulation of skeletal muscle C8 (P = 0.09) and ubiquitin (P = 0.004) mRNA expression occurred post-partum compared with pre-partum. No regulation of hepatic mRNA expression was observed. In conclusion, ubiquitin-mediated proteolysis may contribute to skeletal muscle protein degradation during the periparturient period, and could provide a potential mechanism for attenuation of body protein loss at the onset of lactation.Key words: Lactation, ubiquitin-mediated proteolytic pathway, muscle (skeletal), liver

To characterize the effects of diet and age on the post-absorptive use of fermentation end-products, calves were subjected before and after weaning to plasma glucose, β-hydroxybutyrate and acetate clearance tests. Twenty-four one-week old male Holstein calves were assigned to one of four starter feeds, in a complete random design: (1) control; (2) 10% alfalfa; (3) 20% alfalfa; and (4) cracked corn. Starters were fed ad libitum. Starter intake, carcass-adjusted body weight gain and post-weaning rumen pH were higher in calves consuming alfalfa. Final bodyweights of calves fed alfalfa were 9.5 kg higher than calves given the other diets. With 20% alfalfa, papillae in the caudal ventral blind sac of the rumen were taller than with cracked corn and narrower than on the control. Rumen concentrations of volatile fatty acids were not affected by diet but the acetate:butyrate ratio and pH were higher on d 54 with 20% alfalfa compared to the control. Glucose clearance and flux rates increased significantly from d 11 to 39 but were unaffected to d 53. Increases in plasma β-hydroxybutyrate and acetate concentrations with age were accompanied by corresponding increases in BHB and acetate fluxes, respectively, but no change in the clearance rate constants. There was little effect of diet on clearance of the plasma metabolites. By 8 weeks of age, glucose was cleared from plasma at 2%/min, β-hydroxybutyrate at 16%/min, and acetate at 24%/min. Because of relatively low plasma concentrations of β-hydroxybutyrate and acetate due to incomplete rumen development, glucose remained the predominant energy source for all calves at 8 weeks.

This study examined the localization of cellular glutathione peroxidase (GPx1) and extracellular glutathione peroxidase (GPx3) in lactating mammary tissue and in primary cultures of bovine mammary epithelial cells (BMEC). The effect of selenium as selenomethionine (SeMet) on the growth and viability of BMEC and GPx protein expression and activity were also studied. Single mammary epithelial cells were recovered by serial collagenase/hyaluronidase digestion from lactating bovine mammary tissue and cultured in a low-serum collagen gel system enriched with lactogenic hormones and 0, 10, 20, or 50 nM SeMet. Positive immunostaining with anti-cytokeratin and bovine anti-casein confirmed the epithelial nature and differentiated state of BMEC. Addition of SeMet to media facilitated rapid confluence of BMEC and formation of dome structures. Immunohistochemical and immunocytochemical staining revealed that both GPx1 and GPx3 are synthesized by BMEC and localized in the cytoplasm and nucleus. Up to 50 nM SeMet linearly increased BMEC number and viability over 5 d of culture. Bovine mammary epithelial cells cultured in SeMet-supplemented medium also exhibited markedly elevated GPx activity and linear increases in abundance of GPx1 and GPx3 proteins. It is apparent that SeMet degradation to release Se for synthesis of selenoproteins is carried out by BMEC. Results indicate that bovine mammary epithelial cells express GPx1 and GPx3 in vivo and in vitro; SeMet enhances expression of these selenoproteins in vitro and the growth and viability of BMEC.

The objective of this study was to determine the effects of supplementing glycerol and soybean oil in drinking water on feed and water intake, calculated energy balance, and production performance of periparturient dairy cows. Ninety multiparous Holstein dairy cows were randomly assigned to 1 of 3 treatments: 1) no nutrients supplemented in the drinking water (control); 2) 20 g/L of glycerin supplemented in the drinking water (glycerol); and 3) 10 g/L of soybean oil supplemented in the drinking water (SBO). The trial lasted from 7 d prepartum to 7 d postpartum. Cows were offered a close-up and milking cow TMR for ad libitum intake, pre- and postpartum, respectively. The dry matter intake of cows supplemented with glycerol and SBO was lower than for the control cows throughout the experimental period but not different from each other. Water intake for the control cows was greater than the average for the glycerol and SBO cows prepartum, and greater than for SBO cows but similar to that of glycerol cows postpartum. Glycerol cows consumed more water than SBO cows. There were no differences in energy intake and energy balance of the cows pre- and postpartum. Serum triacylglycerol concentration for glycerol cows was lower than for the control and SBO cows prepartum and was lower than for the SBO cows postpartum. There were no differences in the serum nonesterified fatty acids and glucose concentrations throughout the experiment. There were no differences in the serum beta-hydroxybutyrate (BHBA) concentrations at parturition, but serum BHBA concentration of the glycerol cows was greater than for control and SBO cows during the prepartum period. However, during the postpartum period, serum BHBA concentrations of the control cows were greater than for glycerol and SBO cows. There were no differences in calf birth weights or milk yield and composition. Although the glucogenic property of glycerol supplemented in the drinking water at 20 g/L may not have been sufficient to elicit a milk yield response, it did reduce the concentration of BHBA postpartum.

To test the hypothesis that ammonia detoxification in ruminants consumes amino acids to the detriment of milk protein production, we infused four lactating dairy cows with ammonium acetate or sodium acetate in switchback experiments. Plasma ammonia concentrations increased to 411 μm within 1 h of the start of infusion of ammonium acetate at 567 mmol/h. The rate constant for ammonia clearance from plasma was 0·054/min and the half-life was 12·9 min. Infusion at 567 mmol/h for 1 h followed by 1 h without infusion, repeated four times between am- and pm-milking, caused a decrease in feed intake. Compared with sodium acetate, continuous infusion of ammonium acetate at 360 mmol/h throughout an entire 10-h milking interval increased plasma ammonia concentrations to 193 μm and caused a 20% decrease in milk, protein and lactose production with no effect on percentage composition of milk or the yield of milk fat. Arterial concentrations of glucose and non-esterified fatty acids tended to increase; there was no effect on arterial acetate, β-hydroxybutyrate or triacylglcerol, and branched-chain amino acids, Lys and Thr decreased. Mammary plasma flow, estimated by assuming 100% uptake/output of Phe+Tyr, was significantly correlated with milk yield. Mammary uptakes of acetate tended to be reduced by hyperammonaemia, but uptakes of other energy metabolites and amino acids were not affected. Thus, while an increase in amino acid consumption during hyperammonaemia was apparent from the drop in circulating concentrations of Leu, Ile, Val, Lys and Thr, there was no evidence to support the hypothesis that milk yield is affected by the lower concentrations. An ammonia-induced depression in feed intake may have caused the decrease in milk synthesis.

To identify the relative roles of transla- tion initiation and elongation in the long term control of protein synthesis in ovine tissues, fractional synthe- sis rates (FSR) and ribosomal transit times (RTT) were measured in vivo in 24 ewe lambs at 3 levels of intake (maintenance (M), 1.5M, and 2M) and 8 mature ewes at 2M intake. After 17 to 25 d on treatment, animals were given an i.v. flooding dose of l-(ring-2,6- 3 H)pheny- lalanine and tissues were collected for analysis of ra- dioactivity in free protein, total protein, and nascent ri- bosome-associated proteins. Ribosome transit time (the inverse of elongation rate) averaged 83, 393, 183, 241, 85, and 113 s for liver, duodenum, skin, rumen, semi- membranosus, and LM, respectively. In response to an increased level of intake, protein FSR increased (P 0.50) of intake on RTT in these tissues, and the esti- mated proportion of ribosomes attached to and actively translating mRNA was increased (P < 0.07), indicat- ing that an upregulation of initiation was responsible for the greater FSR. Mature ewes exhibited lower (P < 0.10) protein FSR in all tissues compared with lambs, which was related to a decline in the RNA:protein ratio in all tissues except for liver and duodenum. In all tis- sues but liver and semimembranosus, RTT increased (P < 0.10) with age. The lower elongation rate was not considered to have influenced the protein synthetic rate, but it caused an increase in the proportion of ri- bosomes actively translating mRNA. It is anticipated that this work will provide direction to future studies of the molecular mechanisms of chronic FSR control.

SUMMARYSensitivity analysis is routinely carried out in the evaluation of simulation models to identify the degree to which parameters influence model outputs. This type of sensitivity analysis is much less frequently applied to real systems, but a technique called metabolic control analysis (MCA) was developed in the 1970s for the purpose of experimentally identifying the degree to which individual enzymes in a metabolic pathway influence flux through the pathway. MCA is applied to the results of inhibition, activation or genetic manipulation of enzymatic steps in a biochemical pathway. Flux control coefficients for each enzyme are defined as the fractional change in steady-state flux through the entire pathway for an infinitesimal change in the activity of that one enzyme. The sum of control coefficients in a linear, non-branching pathway is equal to one. It is a common finding in MCA that the control, or sensitivity, is distributed over multiple enzymes and not in a single rate-limiting enzyme. The fundamental principles of MCA are reviewed and an overview of experimental methods to measure control coefficients is provided, with the objective of introducing this approach to the fields of agricultural biochemistry and modelling, where it is little known. The application of MCA to the study of glucose metabolism and fatty acid synthesis in bovine mammary tissue are reviewed. The analyses indicated that mammary hexokinase activity exerts more control than transmembrane transport of glucose over lactose synthesis, and that control of cytosolic fatty acid synthesis is shared between acetyl-CoA carboxylase and fatty acid synthase, contrary to the widely held view that acetyl CoA carboxylase is the rate-limiting enzyme. It is suggested that MCA could be a valuable aid in the integration of proteomic and metabolomic data with metabolic flux measurements to engineer desired changes in the composition of milk from dairy animals.

To establish the duration of a severe feed restriction necessary to reversibly decrease milk production in high-producing Holstein cows, milk, plasma and urine parameters were assessed every 6 h during a 24-h restriction and 18-h refeed period. The 24-h restriction decreased yields of milk, protein and lactose by more than 30% without inducing ketosis or impairing milk production post-trial. Key words: Feed restriction, dairy cows, milk yield, plasma metabolites

Experiments were conducted to investigate the use of added flavor in drinking water of Holstein calves and lactating dairy cattle to determine effects on dry feed intake. Nine calves were used in a replicated 3 x 3 Latin square design, and water offered was unflavored or flavored with orange or vanilla. All calves were offered commercial starter. Feed intake of the dry starter was increased in calves offered the orange flavor treatment compared with the control or the vanilla treatment. The increased dry feed intake agreed with the significant increase in weight gain measured in calves on the orange treatment. Further experiments were performed with 4 second-lactation cows using the addition of another orange flavor to the water compared with unflavored water under conditions of free access or time-restricted water access. No significant changes were found for dry matter intake, water consumption, or milk yield. These findings demonstrate an important finding that flavoring agents need not be added only to the starter feed for calves, but flavor can stimulate dry feed intake and BW gain when used in drinking water.

To determine how glucose modulates protein synthesis when essential AA are in abundant supply, 5 early-lactation, rumen-fistulated Holstein dairy cows were fed a diet containing 6.95 MJ/kg of net energy for lactation and 12.4% crude protein and abomasally infused for 5 d with saline, 844 or 1,126 g/d of a complete essential AA mix, with and without the inclusion of 1,000 g/d of glucose, in a 5×5 Latin square design. Infusion of essential AA increased milk yield by 4.1 kg/d, milk protein by 256 g/d, milk fat by 95 g/d, and milk urea nitrogen by 70% compared with saline, with no differences between the level of essential AA infusion. The addition of glucose to essential AA infusate did not stimulate milk protein yield or concentration, but reduced milk urea nitrogen by 17% and decreased milk fat yield. Arterial concentrations of total essential AA increased 3- to 4-fold, mammary clearance decreased 61%, and mammary uptake of essential AA increased 65% in response to essential AA infusion. Arterial branched-chain AA concentrations declined 29% in response to glucose and mammary clearance increased 48%, but mammary AA uptake was unchanged. Essential AA infusion increased plasma 3-methylhistidine by 50% and reduced muscle branched-chain α-keto acid dehydrogenase kinase abundance by 14%, indicating stimulation of muscle protein turnover and branched-chain AA catabolism, respectively. Glucose had no further effect on muscle branched-chain α-keto acid dehydrogenase kinase abundance but decreased mRNA expression of branched chain aminotransferase 1. Lack of further increases in plasma 3-methylhistidine or greater stimulation of muscle branched-chain AA catabolism indicates that muscle protein degradation was unchanged with glucose but that accretion may have been stimulated. The decrease in circulating branched-chain AA concentrations and nitrogen excretion in response to glucose suggests that surplus essential AA were redirected to peripheral, extra-mammary tissues.

Deficiencies and imbalances of specific group II essential amino acids (EAA) were created in lactating cows by an infusion subtraction protocol to explore effects on milk production and abundance and phosphorylation state of regulators of mRNA translation in the mammary glands. Five lactating cows on a diet of 11.2% crude protein were infused abomasally for 5d with saline, 563 g/d of a complete EAA mix, or EAA mixes without the branched-chain amino acids (BCAA), Leu, or Lys in a 5 × 5 Latin square design. Milk protein yield was stimulated by EAA infusion and returned to saline levels upon subtraction of BCAA, Leu, or Lys. Mammary abundance of phosphorylated S6K1 was measured as an indicator of mammalian target of rapamycin complex 1 (mTORC1) activity and was found not to be affected by the complete EAA mix but was increased by the mixture lacking Lys. Total S6K1 abundances in mammary tissue were elevated by complete and BCAA-lacking infusions. All of the EAA treatments except the one lacking BCAA upregulated mammary eIF2Be and eIF2α abundances, which is stimulatory to global mRNA translation. Phosphorylation state of eIF2Be tended to decrease when complete or Lys-lacking EAA mixtures were infused. Phosphorylation state of eIF2α was not affected by treatment. We detected a correlation of 0.62 between phosphorylation state of S6K1 and total eIF2Be abundance, and a correlation of 0.58 between phosphorylation state of S6K1 and total eIF2α abundance, suggesting that mTORC1 activation may have upregulated eIF2Be and eIF2α expression. Despite maintenance of mammary eIF2Be and eIF2α abundances during Leu and Lys deficiencies, milk protein yield declined, suggesting that other factors are responsible for mediating effects of Lys and Leu. A deficiency of all 3 BCAA may impair milk protein yield through deactivation of mTORC1-mediated upregulation of eIF2Be and eIF2α abundances.

The rate of secretion of αs2-casein into bovine milk is approximately 25% of that of β-casein, yet mammary expression of their respective mRNA transcripts (csn1s2 and csn2) is not different. Our objective was to identify molecular mechanisms that explain the difference in translation efficiency between csn1s2 and csn2. Cell-free translational efficiency of csn2 was 5 times that of csn1s2. Transcripts of csn1s2 distributed into heavier polysomes than csn2 transcripts, indicating an attenuation of elongation and/or termination. Stimulatory and inhibitory effects of the 5′ and 3′ UTRs on translational efficiency were different with luciferase and casein sequences in the coding regions. Substituting the 5′ and 3′ UTRs from csn2 into csn1s2 did not improve csn1s2 translation, implicating the coding region itself in the translation difference. Deletion of a 28-codon fragment from the 3′ terminus of the csn1s2 coding region, which displays codons with low correlations to cell fitness, increased translation to a par with csn2. We conclude that the usage of the last 28 codons of csn1s2 is the main regulatory element that attenuates its expression and is responsible for the differential translational expression of csn1s2 and csn2.

Previous work shows that mammary uptake of milk precursors from blood can be affected by the rate of blood flow (F) to the glands. The purpose of the current work was to test the ability of compartmental and cylindrical capillary models to account for the variation in mammary extraction and net uptake of plasma metabolites produced by perturbation of mammary F. The data for model fitting were obtained from a previous experiment in which mammary arteriovenous differences of acetate + β-hydroxybutyrate (2C), glucose, triacylglycerol (TAG), and long-chain fatty acids (LCFA) were measured in 4 cows before, during, and after intraarterial infusion of inhibitors of endothelial nitric oxide synthase and cyclooxygenase, which are 2 major systems of F control in the mammary glands. The 4 models tested were (1) constant extraction within each cow, (2) clearance from an extracellular compartment is a linear function of F with an intercept, (3) total capillary volume in a cylindrical representation is a linear function of F with an intercept, and (4) uptake from an extracellular compartment obeys Henri-Michaelis-Menten kinetics, where maximum velocity (Vmax) is a linear function of F with an intercept. According to prediction errors, model 4 fitted 2C extraction data best, accounting for 82% of the observed variation. The estimated Km (Henri-Michaelis-Menten constant) for venous 2C was 0.4 mM. For glucose clearance, a variant of model 2 with a positive effect of 2C uptake on clearance was identified as best, producing a coefficient of determination (R(2)) of 0.31. For TAG, model 2 with a positive effect of arterial TAG concentration on TAG clearance was best, with an R(2) of 0.22. For LCFA, model 2 with a positive effect of arterial LCFA on LCFA clearance was best, with an R(2) of 0.29. Models 2 and 3 fitted the extraction data with the same R(2)-values and prediction errors, so both compartmental and cylindrical approaches to describing the vascular bed were equally capable of describing the effect of F on mammary uptakes. A combined fit of all best-fit models to extraction data for all 4 metabolites at once explained 52, 42, 73, and 77% of variation in net uptakes of 2C, glucose, TAG, and LCFA, respectively. According to the fitted model, each 1 L/min increase in F increased the mammary volumes of distribution of 2C, glucose, TAG, and LCFA by 13, 14, 18, and 7%, respectively.

The objectives of this research were to determine the flux control coefficients for acetyl CoA carboxylase and fatty acid synthase using an in vitro preparation of bovine mammary homogenate. For an enzyme to be considered rate limiting with the use of metabolic control analysis, its control coefficient would be equal to unity. The hypothesis for this experiment was that the control coefficient for acetyl CoA carboxylase was not equal to unity, and that this enzyme was not, therefore, the rate-limiting step. Mammary tissue was isolated from lactating Holstein cows at slaughter and frozen in liquid nitrogen. Tissue was ground, homogenized, and centrifuged to obtain a postmitochondrial supernatant for use in in vitro incubations containing labeled acetate. Specific inhibitors for acetyl CoA carboxylase and fatty acid synthase were used to fractionally inhibit de novo synthesis for the calculation of flux control coefficients. The composition of fatty acids synthesized in the absence of enzyme inhibitors was similar to the composition of fatty acids in the presence of inhibitors. Calculations following avidin inhibition of acetyl CoA carboxylase determined the flux control coefficient was 0.63 +/- 0.15, which means that 63% of the control of fatty acid synthesis is exerted by acetyl CoA carboxylase. The remaining control (37%) was from fatty acid synthase, which indicates a significant degree of control over the flux of acetate in de novo synthesis resides with this enzyme. The rate-limiting status ascribed to acetyl CoA carboxylase was not supported, because the flux control coefficient was less than unity. Metabolic control analysis, through its use of pathway product measurements, allows for potential interactions in the pathway such as feedback inhibition contribution to the flux control coefficients, which would not otherwise be considered in studies measuring enzyme kinetics with purified enzymes.

Lactating cows were exposed to large amino acid imbalances and deficiencies by i.v. infusion to characterize responses in milk production and plasma concentrations of metabolites and hormones. Six cows in early lactation were fed a basal diet of 9% CP and infused continuously for 6 d with saline (negative control), 1.1 kg/d of a complete amino acid mix (positive control), or the equivalent mix lacking Met, Lys, His, or all 3 branched-chain amino acids. All cows received all treatments in 6 successive periods in a Latin square design. Infusion of the complete amino acid mix resulted in an increase in the plasma concentrations of several essential amino acids, insulin, and glucagon. Milk protein production was stimulated by 19%, which accounted for 10% of the infused amino acid. Plasma urea, acetate, and beta-hydroxybutyrate concentrations were increased. Compared with saline, the amino acid mixtures lacking Met, Lys, or His increased essential amino acids, glucose, insulin, and glucagon concentrations in plasma, and decreased growth hormone. Plasma concentration of the essential amino acid absent from the infusate fell 2-fold but milk protein yield remained within 12% of its basal value. Dry matter intakes were depressed 35% over the first 2 d of infusion of imbalanced mixtures but recovered thereafter. Milk fat yields were increased 258 and 320 g/d by mixtures devoid of Lys and His, respectively. Correction of a Met, Lys, or His deficiency did not affect hormone concentrations in plasma and milk protein yield increased 27% due entirely to increased concentration of the single amino acid in plasma. Although imbalance and deficiency generated similar amino acid profiles in plasma, it was concluded that endocrine responses to total amino acid supply during imbalance can override imperfections in the circulating amino acid profile to maintain milk protein yield at higher levels than expected from deficiency states. Both imbalance and deficiency were characterized by a low protein:fat ratio in milk. Infusion of a mix of amino acids lacking Val, Ile, and Leu, despite a decrease in plasma Leu to 58% of its basal value, increased milk protein and fat yields to the same extent as the complete amino acid mix.

In the dynamic modeling of dairy cow performance over a full lactation, the difference between net energy intake and net energy used for maintenance, growth, and output in milk accumulates in body reserves. A simple dynamic model of net energy balance was constructed to select, out of some common dry matter intake (DMI) prediction equations, the one that resulted in a minimum cumulative bias in body energy deposition. Dry matter intake was predicted using the Cornell Net Carbohydrate and Protein System, Agricultural Research Council, or National Research Council (NRC) DMI equations from body weight (BW) and predicted fat-corrected milk yield. The instantaneous BW of cows at progressive weeks of lactation was simulated as the numerical integral of the BW change obtained from the predicted net energy balance. Predicted DMI and BW from each DMI equation, using either of 2 equations to describe maintenance energy expenditures, were compared statistically against observed data from 21 herd average published full lactation data sets. All DMI equations underpredicted BW and DMI, but the NRC DMI equation resulted in the minimum cumulative error in predicted BW and DMI. As a general solution to prevent predicted BW from deviating substantially over time from the observed BW, a lipostatic feedback mechanism was integrated into the NRC DMI equation as a 2-parameter linear function of the relative size of simulated body reserves and week of lactation. Residual sum of squares was reduced on average by 52% for BW predictions and by 41% for DMI predictions by inclusion of the negative feedback with parameters taken from the average of all 21 least squares fits. Similarly, root mean square prediction error (%) was reduced by 30% on average for BW predictions and by 23% for DMI predictions. Inclusion of a feedback of energy reserves onto predicted DMI, simulating lipostatic regulation of BW, solved the problem of final BW deviation within a dynamic model and improved its DMI prediction to a satisfactory level.

Equations that predict daily dry matter intake (DMI) of a lactating cow could be evaluated by comparing the predicted accumulation of energy in body weight (BW) over the course of lactation with the observed BW evolution. However, to do so requires that first the energy balance calculations from observed DMI are evaluated. The purpose of the work reported here was to determine the degree of deviation of predicted from observed BW, according to net energy for lactation (NE(L)) balance calculated from weekly observations of DMI, BW, and fat-corrected milk production in 21 sets of full-lactation data, and to determine an appropriate correction of the NE(L) bias for subsequent DMI prediction evaluations. When the National Research Council maintenance equation 0.08 x BW(kg)(0.75) was used in energy balance calculation, BW was overpredicted with an increasing difference between the cumulative predicted BW and observed BW as lactation progressed. Placing all the error of BW prediction into maintenance energy expenditures resulted in a best-fit equation of 0.096 +/- 0.003 Mcal/kg of BW(0.75). A time-dependent equation was also developed, in which weekly maintenance expenditures were determined as the NE(L) expenditure to yield a zero NE(L) balance and could be described by a second-order polynomial equation related to week of lactation (WOL) where maintenance NE(L) = [-0.0227(+/- 0.0098) x WOL2 + 1.352(+/- 0.456) x WOL + 78.09(+/- 4.92) Mcal/kg of BW(0.75)] x 10(-3). Average maintenance energy expenditure at the onset of lactation was approximately 0.08 Mcal/kg of BW(0.75), and this value increased to a plateau at wk 15 of lactation of approximately 0.098 Mcal/kg of BW(0.75). Standard deviations between data sets of weekly maintenance parameter estimates throughout lactation were large but consistent at approximately 25% of the mean. Revision of the maintenance energy expenditure estimate substantially improved BW prediction by the energy balance model. On average, the 0.096 Mcal of NE(L)/kg of BW(0.75) equation resulted in the best BW predictions, although substantial variation existed around this value.

Thirty-three paired indicator/nutrient dilution curves across the mammary glands of four cows were obtained after rapid injection of para-aminohippuric acid (PAH) plus glucose into the external iliac artery. For the measurement of extracellular volume and kinetics of nutrient uptake from indicator dilution curves, several models of solute dispersion and disappearance have been proposed. The Crone-Renkin models of exchange in a single capillary assume negligible washout of solutes from the extracellular space and do not describe entire dilution curves. The Goresky models include a distribution of capillary transit times to generate whole system outflow profiles but require two indicators to parametize extracellular behavior. A compartmental capillary, convolution integration model is proposed that uses one indicator to account for the extracellular behavior of the nutrient after a paired indicator/nutrient injection. With the use of an iterative approach to least squares, unique solutions for nonexchanging vessel transit time μ and its variance σ were obtained from all 33 PAH curves. The average of heterogeneous vascular transit times was approximated as 2σ = 8.5 s. The remainder of indicator dispersion was considered to be due to washout from a well-mixed compartment representing extracellular space that had an estimated volume of 5.5 liters or 24% of mammary gland weight. More than 99% of the variation in the time course of venous PAH concentration after rapid injection into the arterial supply of the mammary glands was explained in an unbiased manner by partitioning the organ into a heterogeneous nonexchanging vessel subsystem and a well-mixed compartmental capillary subsystem.

The effect of dietary P content on P balance was evaluated using 14 lactating dairy cows fed a corn silage based total mixed ration (TMR) containing either 0.24% P or 0.36% P. The animals were paired by calving date at dry-off with one cow from each pair randomly assigned to either dietary treatment. All cows were fed a common TMR containing 0.34% P prepartum, and then 0.41% P for 2 wk postpartum. Experimental diets were fed from 3 to 13 wk postpartum. Phosphorus balance was conducted at week 13 postpartum. Cows fed 0.36% P diet consumed more (P < 0.05) P than the 0.24% P diet cows (69.2 vs. 50.9 g d-1) and excreted more (P < 0.05) P in the faeces (49.1 vs. 25.7 g d-1). Phosphorus digestibility was lower (P < 0.05) for the 0.36% P diet than the 0.24% P diet (29.0 vs. 48.8%). Dry matter intake and milk yield were not (P > 0.05) affected by treatments. These results suggest that short-term P balance in lactating dairy cows can be maintained on a diet supplying 26% below National Research Council 2001 recommendations of P while reducing the amount of P excreted into the environment. Key words: Phosphorus balance, dairy cows, lactating, phosphorus requirement

To quantify kinetics of mammary glucose utilization in vivo, 24 paired glucose and extracellular indicator ( p-aminohippuric acid) dilution curves across intact bovine mammary glands were obtained after bolus injections into the external iliac artery. Dilution curves were analyzed using a compartmental capillary, convolution integration model. Four candidate submodels of glucose transport and metabolism in capillary supply zones were fit to the glucose dilution curves and evaluated. Model I, with one extracellular compartment for glucose and first-order unidirectional uptake, failed, indicating that efflux of glucose from the intracellular space could not be ignored. Model II, with first-order exchanges between extracellular and intracellular compartments and sequestration from the latter, was overdefined because unidirectional clearance of glucose was at least five times the blood flow rate and 20 times the net clearance rate. Model III, combining extracellular and intracellular space into one compartment, was superior in its goodness-of-fit to curves and identifiability of parameters. Michaelis-Menten parameters of sequestration were not identifiable. Parameters of the optimal compartmental capillary, convolution integration model were applicable to both the dynamics of injected glucose dilution and the steady-state background arteriovenous difference of glucose. Glucose sequestration followed first-order kinetics between 0 and 7 mM extracellular glucose with an average rate constant of 0.006 s−1 or a clearance of 44 ml/s. The ratio of intracellular to extracellular glucose distribution space was 0.34, which is considerably lower than the expected intracellular volume and suggests an intracellular occlusion compartment with which extracellular glucose rapidly exchanges.

A computer model was developed to predict responses of lactating ewes to concentrate supplementation, whether on pasture or stall-fed, given concentrate once per day or in multiple feedings, and suckling multiple lambs. The model considers effects of concentrate supplementation on organic acid production, saliva flow, ruminal pH, and forage intake. The user defines ewe BW, feed composition, and concentrate feeding times and amounts. The reference ewe has free access to forage and water. Upon consumption, forages and concentrates enter into lag pools for 2.0 and 0.24 h, respectively. Carbohydrates then enter ruminal pools of degradable fiber, undegradable fiber, or nonstructural carbohydrate, from which they are degraded or pass to the lower gut. Rapid dissociation of organic acids from carbohydrate fermentation and buffers from rumination are simulated to determine ruminal pH according to the Henderson-Hasselbach equation. The pH, in turn, affects fiber degradation rates. Forage intake continues during daylight hours until ruminal NDF exceeds 1.0% of BW, or organic acid concentration exceeds 130 mM. A circadian pattern of organic acid concentrations and pH of rumen contents with multiple concentrate feedings was simulated by the model with root mean square prediction error of 7.7 and 3.0 to 4.0% of the observed mean, respectively. However, ignoring fermentation of dietary protein may have caused an underestimation of organic acid production rates. The model predicted the increase in total DMI and the substitution effect on forage intake of increasing levels of concentrate supplementation. Simulations suggested that a single concentrate meal daily was best fed in the evening to minimize the substitution effect, and that there was no benefit in forage intake to feeding 2 kg/d concentrate in more than two meals per day.

Our objectives were to examine the effect of an i.p. injection of a flooding dose of L-phenylalanine (Phe) containing L-(ring- 2 H5)Phe on time courses of physiologic responses, the tracer Phe enrichments, and fractional protein synthesis rates (FSR) in plasma, visceral organs, and muscles. In a randomized complete block design, 5 blocks of 5 littermate piglets were weaned at 16 d of age and injected i.p. with a flooding dose of L-Phe (1.5 mmol/kg body weight) on d 8 postweaning under fed conditions. Tissues were collected at 15, 30, 45, 60, and 75 min postinjection. Plasma glucose concentration increased (cubic effect, P 0.05) from 4.8 preinjection to 5.8 mmol/L 15 min postinjection and returned to preinjection levels thereafter. Plasma insulin concentration did not change (P 0.05) over time. Plasma Phe concentration increased logarithmically (P 0.05) from 85 to 711 mol/L and reached 95% of the maximum concentration 48 min postinjection, but no changes (P 0.05) in tissue contents of other free amino acids were observed. The Phe free pools in plasma, visceral organs, and muscles were evenly enriched (32.3 1.4 mol%) with L-( 2 H5)Phe 15 min after the i.p. injection. The FSR in visceral organs did not change (P 0.05), whereas plasma and muscle protein FSR decreased (P 0.05) over time. We conclude that the i.p. injected tracer Phe rapidly distributed into plasma and intra- and extracellular spaces, and was effective for measuring FSR in visceral organs, but not in plasma and muscles of pigs. J. Nutr. 134: 2722-2728, 2004.

The excretion of major odor-causing and acidifying compounds in response to dietary supplementation of chicory inulin extract was investigated with six Yorkshire barrows, with an average initial BW of 30 kg, according to a balanced two-period cross-over design. The animals were fed a control diet containing no inulin extract and a treatment diet with 5% inulin extract (as-fed basis) at the expense of cornstarch. Each diet was formulated (as-fed basis) to contain 16% CP from corn (51%) and soybean meal (29%). Each experimental period lasted 14 d, with 10 d for dietary adaptation and 4 d for collection of fecal and urine samples. The fecal samples were analyzed for four major classes of odor-causing and acidifying compounds: 1) VFA; 2) N-containing compounds, including total N and ammonia; 3) volatile sulfides measured as hydrogen sulfide units; and 4) phenols and indoles, including p-cresol, indole, and skatole. Supplementation of chicory inulin at 5% had no effects on the fecal excretion of VFA (P = 0.29), ammonia (P = 0.96), total volatile sulfides (P = 0.56), p-cresol (P = 0.56), and indole (P = 0.75). Fecal excretion of total N (inulin = 6.13 vs. control = 5.10 g/kg DMI) was increased (P < 0.05), whereas urinary total N excretion (inulin = 15.1 vs. control = 16.4 g/[pig x d]) was not affected (P = 0.17) by the inulin supplementation compared with the control group. Furthermore, fecal excretion of skatole (inulin = 9.07 vs. control = 18.93 mg/kg DMI) was decreased (P < 0.05) by the inulin supplementation compared with the control group. In conclusion, dietary supplementation of 5% chicory inulin extract is effective in decreasing the fecal excretion of skatole in growing pigs fed corn and soybean meal diets.

Initial rates of glucose entry into isolated bovine mammary epithelial cells display moderate degrees of asymmetry and cooperative interactions between export and import sites. The present study examined the hypothesis that these kinetic features are due to compartmentalization of intracellular glucose. Net uptake of 3- O-methyl-d-[1-3H]glucose (3-OMG) by isolated bovine mammary epithelial cells was measured at 37°C. The time course of 3-OMG net uptake was better fitted by a double-exponential equation than by a single- or triple-exponential equation. Compartmental analysis of the time course curve suggested that translocated 3-OMG is distributed into two compartments with fractional volumes of 32.6 ± 5.7% and 67.4 ± 5.7%, respectively. The results support the view that glucose transport in bovine mammary epithelial cells is a multistep process consisting of two serial steps: fast, carrier-mediated, symmetric translocation of sugar across the cell plasma membrane into a small compartment and subsequent slow exchange of posttranslocated sugar between two intracellular compartments. A three-compartment model of this system successfully simulated the observed time course of 3-OMG net uptake and the observed dependence of unidirectional entry rates on intra- and extracellular 3-OMG concentrations. Simulations indicated that backflux of radiolabeled sugar from the small compartment to extracellular space during 15 s of incubation gives rise to the apparent asymmetry, trans-stimulation, and cooperativity of mammary glucose transport kinetics. The fixed-site carrier model overestimated the rate of glucose accumulation in cells, and its features can be accounted for by the compartmentalization of intracellular sugar.

An experiment was conducted to examine effects of feeding either fresh alfalfa or alfalfa silage on milk fatty acid profiles in Holstein dairy cows. Sixteen multiparous cows (664.2±65.7 kg body weight; 187±56 days in milk) were used in a crossover design with two 5-week periods, with measurements being taken during week 5 of each period. Feed intake was higher (P=0.03) in cows consuming alfalfa silage compared with those cows fed fresh alfalfa. Milk yield and milk components were not affected (P>0.05) by dietary treatment. Proportions of myristic (C14:0) and palmitic (C16:0) acids were lower (P