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Essential amino acid (EAA) deficiencies and imbalances were created in lactating cows by using an infusion subtraction protocol to explore effects on milk protein yield and activity state of regulators of mRNA translation in the mammary glands. Six lactating cows on a diet of 11.2% protein were infused abomasally for 5 d with saline, 563 g/d of a complete EAA mix, or EAA without His, Met, Phe, or Trp in a 6 × 6 Latin square design. Infusion of complete and imbalanced EAA solutions increased mammalian target of rapamycin (mTOR) signaling in the mammary glands, as evidenced by higher ribosomal S6 kinase 1 (S6K1) phosphorylation compared with saline infusion. Total S6K1 abundance was decreased by imbalanced AA infusions. Except for the mixture lacking Phe, infusion of EAA, whether imbalanced or not, increased abundance of total eukaryotic initiation factor 2Be (eIF2Be). A correlation of 0.33 between phosphorylation state of S6K1 and total eIF2Be abundance suggests that an mTOR-mediated upregulation of eIF2Be translation occurred. Despite increased mTOR/eIF2Be signaling, milk protein yields increased only with the complete EAA mixture compared with saline. Low plasma concentrations of His, Met, and Phe during their respective imbalances likely interfered with protein synthesis. Total abundance and phosphorylation state of eukaryotic initiation factor 2α were not responsible for the interference. Further study of eIF2Be as a regulator of milk protein yield is warranted.

Background Decreasing the rate of protein emptying from the stomach may improve efficiency of utilization of dietary amino acids for protein deposition. Some studies in rats and humans have shown casein to be more slowly released from the stomach than whey protein. To test if casein induces a slower rate of gastric emptying in cats than whey protein, L-[1-13C]phenylalanine (Phe) was dosed orally into 9 adult cats to estimate gastric emptying and whole-body Phe flux. Results Concentrations of indispensable amino acids in plasma were not significantly affected by dietary protein source. First-pass splanchnic extraction of Phe was not different between diets and averaged 50% (SEM = 3.8%). The half-time for gastric emptying averaged 9.9 min with casein and 10.3 min with whey protein, and was not significantly different between diets (SEM = 1.7 min). Phenylalanine fluxes were 45.3 and 46.5 μmol/(min · kg) for casein- and whey-based diets, respectively (SEM = 4.7 μmol/(min · kg)). Conclusions In adult cats fed frequent small meals, the replacement of casein with whey protein in the diet does not affect supply or utilization of amino acids. These two milk proteins appear to be equally capable of meeting the dietary amino acid needs of cats.

To test the effect of mammary blood flow on net uptakes of milk precursors by the mammary glands, inhibitors of nitric oxide synthase (NOS) and cyclooxygenase (COX) were infused into the mammary circulation of 4 lactating cows. Inhibitors were infused in a 4×4 Latin square design, where treatments were infusion for 1 h of saline, NOS inhibitor (N ω -nitro-l-arginine methyl ester hydrochloride), COX inhibitor (indomethacin), or both NOS + COX inhibitors into one external iliac artery. Para -aminohippuric acid was also infused to allow for estimation of iliac plasma flow (IPF), of which approximately 80% flows to the mammary glands. Blood samples were collected before, during, and after inhibitor infusion from the contralateral external iliac artery and ipsilateral mammary vein. Inhibition of COX and NOS each produced a decrease in IPF, although the NOS effect was smaller and IPF continued to be depressed throughout the recovery period. The combination of COX and NOS inhibition produced a 50% depression in IPF and there was no carryover into the recovery period. Treatments that depressed IPF also increased arterial concentrations of acetate, β-hydroxybutyrate (BHBA), and glucose. Similarly, arteriovenous differences of acetate, BHBA, and glucose were all increased during IPF depression. To correct for a potential effect of arterial concentration, arteriovenous differences were normalized to arterial concentration, producing an extraction percentage. Inhibition of COX increased glucose extraction and tended to increase acetate and BHBA extraction. Dual inhibition only increased BHBA extraction and had no effect on mammary extraction of other metabolites. These extractions did not increase because clearances of glucose and TAG decreased as IPF decreased, and clearances of acetate and BHBA tended to decrease. Net uptake of TAG was depressed by dual NOS/COX inhibition, whereas uptakes of acetate, BHBA, and glucose were not affected by any of the treatments. To separate effects of flow from effects of arterial concentration, uptakes were regressed against IPF and arterial concentration simultaneously. According to the slopes of the regressions, a 10% decrease in IPF from the mean observed during saline infusion resulted in 3.8, 7.3, and 10.4% decreases in uptakes of acetate, glucose, and triacylglycerol, respectively. These findings indicate that mammary blood flow affects milk precursor uptake, and that clearance should not be assumed constant to predict mammary uptakes of milk precursors in situations where blood flow is changing.

The objective of this study was to determine the effect of enhanced whole-milk (WM) feeding systems in calves from birth to 8wk of age on subsequent first-lactation performance. The experiment was conducted as a completely randomized design consisting of 2 treatment groups. At birth, 152 Holstein heifer calves were randomly assigned to 1 of 2 treatments: (i) 4L of WM/d or (ii) 8L of WM/d. The calves were bucket fed 2 or 4L of WM twice daily at 0700 and 1600h. Each calf was housed individually in temperature-controlled nurseries and had ad libitum access to water and textured calf starter daily. Calves consumed greater volumes of textured calf starter when fed 4 versus 8L of WM/d. Water intakes mirrored starter intakes, leading to greater water consumption at weaning. Calves reared on 8L of WM/d were heavier at d 56 than calves reared on 4L of WM/d. The average daily gain of the calves offered 8L of WM/d from d 0 to 56 was greater than that of calves offered 4L of WM/d. Structural measurements were significantly greater for calves that consumed 8L of WM/d. The differences observed in withers height and live BW due to WM feeding level were not apparent by 3 and 12mo of age, respectively. Rumen pH was higher in calves that consumed 8L of WM/d than in calves that consumed 4L of WM/d. Whole-milk feeding level did not affect age at first calving or milk-production parameters. These results suggest that enhanced WM feeding improved growth performance until 3mo of age. However, first-lactation results indicated no lactation-performance benefits of increased nutrition and growth performance during the milk-fed period in dairy calves.

To evaluate a close mammary infusion technique for the study of milk protein responses to blood amino acid profile, five early-lactation, multiparous Holstein cows were surgically fitted with catheters in both external iliac arteries. Animals were infused into one arterial catheter with five different solutions on 5 consecutive days in a Latin square design. Infusions began at 0800 h and continued until 1800 h. The five infusates were a 3% saline control, 15 g/h of complete amino acid mix, 15 g/h of imbalanced amino acid mix (minus His), 30 g/h of complete amino acid mix, and 30 g/h of imbalanced amino acid mix (minus His). Cows were fed a total mixed ration twice daily containing 16% crude protein and 1.7 Mcal/kg of net energy for lactation. Infusion of the complete amino acid mix elevated amino acid concentrations in arterial plasma two- to threefold but caused only a small dose-dependent increase in milk protein content and yield. Fat percentage in milk was decreased from 4.08 to 3.35% by the complete amino acid infusions so that the protein:fat ratio climbed from 0.76 on the control to 0.99 with 30 g/h of amino acid. Removal of His from the infusate caused plasma His concentrations to drop but had no effect on any other circulating amino acids. Milk composition was restored to control levels by removal of the single amino acid. A short-term circulating amino acid imbalance depresses milk protein percentage and increases milk fat content in dairy cows.

A kinetic modelling approach was developed and investigated with the aim of predicting the utilization of major substrates in the mammary gland and milk secretion rates in the lactating cow at varying concentrations of substrate in arterial blood. The model includes kinetic equations of transport and metabolism of glucose, acetate, free amino acids and free fatty acids in secretory cells and a phenomenological description of autoregulation of local blood flow, in which an energy criterion of control has been used. The predicted relationships between the rate of milk secretion and glucose levels in the blood are consistent with experimental results. Differential stimulation of α-lactalbumin synthesis causes increments in local blood flow and milk secretion rate in the model. The results of the study suggest that there is no simple relationship between the level of substrates in the blood and milk yield and contents of fat and protein in milk. This is because the effect on production of varying patterns of substrate concentrations in the blood is mediated by network interactions at the level of secretory cell metabolism and microcirculation. However, dynamic modelling provides a rational framework for developing such predictive tools.

To test the hypothesis that the uptake capacity of the bovine small intestine for glucose is upregulated to match or slightly exceed glucose delivery, glucose was continuously infused into the proximal duodenum of four cannulated holstein heifers. Every 3 days, infusion rates were increased by an average of 34 mmol/h. A model of glucose disappearance from multiple boluses of intestinal digesta was used to estimate the transporter maximum velocity and functional maximum uptake capacity for the entire small intestine from average ileal glucose flows during the third day of each period. Because of its intermittency, digesta flow remained independent of simulated transit time. For each unit increase in glucose infusion rate, uptake capacity increased by only 0.55 units. Excess capacity for glucose uptake was approximately twofold in forage-fed cattle and declined to below delivery at infusions of >208 mmol/h added glucose, approximately three times the normal load. Calculations for cattle, sheep, and rats indicate that the glucose transport capacity of the small intestine is typically underutilized because of a fraction of time that transporters are not in contact with digesta.

Four primparous Holstein cows were used in a 4 × 4 Latin square with 21-d periods to determine the effect of redfish oil and monensin sodium on milk composition. The four dietary treatments were a basal diet (control), the basal diet plus 14.5 mg monensin kg−1 dietary dry matter (M), 2% fish oil (FO), and a combination of fish oil and monensin (FO + M). Total DM intake, measured during the last week of each period was reduced on the two fish oil treatments but an interaction with monensin depressed intakes further. An additive inhibition of rumen fibre degradation is discussed. Differences in yields of milk and lactose were not shown to be significant among treatments. Milk fat content was reduced 29.8% by fish oil supplementation and protein content dropped 5.3%. Protein:fat ratios increased from 0.78 on the control ration to 1.08 on the fish oil treatments. Monensin, on the other hand, only caused a change in milk fat percentage, and that by a 7.5% decline. Fish oil increased the concentrations of 20- and 22-carbon fatty acids in milk, including the n-3 eicosapentaenoic and docosahexaenoic acids which were transferred at 9.3 and 16.2% efficiency from the diet, respectively. Monensin had no effect on milk fatty acid profile. The fish-oil-by-monensin interaction produced the poorest energy intakes and rates of energy deposition into milk. Key words: Milk composition, fish oil, monensin, omega-3 fatty acids

The objective of this study was to determine effects of glucose on milk protein yield and mammary mammalian target of rapamycin (mTOR) activity in dairy cattle in early lactation. Eight multiparous cows at 73 ± 8 d in milk were randomly assigned to 2 treatments in a crossover design for two 6-d periods. Treatments were jugular infusion of either saline (Sal) or 896 g/d glucose (Glc). All cows were fed a total mixed ration with 42% neutral detergent fiber, had free access to water, and were milked twice a day. Within each period, blood samples were taken (d 5) and mammary tissue was collected by biopsy (d 6) from each hindquarter for Western blot analysis. In addition to Sal and Glc treatments, on d 6, rapamycin dissolved in 50% dimethyl sulfoxide was administered via the teat canals into the left quarters, with a control solution administered into the right quarters. Rapamycin had no effect on milk protein yields or phosphorylation state of mTOR signaling proteins. Infusions of Glc significantly increased milk yield but only tended to increase milk protein yields. Milk fat tended to be decreased in cows infused with Glc, whereas lactose yields were significantly increased. Glucose infusion did not increase plasma glucose levels, but insulin and nonessential AA concentrations increased by 21 and 16%, respectively, branched-chain AA concentrations decreased 24%, and essential AA concentrations tended to decrease by 14%. Infusion of Glc significantly decreased abundances of both phosphorylated and total ribosomal S6 kinase 1 (S6K1) in mammary tissue by 27 and 11%, respectively. Abundance of phosphorylated eukaryotic initiation factor 4E-binding protein 1 (4EBP1) decreased significantly by 25%, whereas total 4EBP1 exhibited a tendency to decrease by 16%. We conclude that the mTOR signaling pathway is not the only regulator of milk protein synthesis. Decreases in essential AA concentrations in plasma suggest that protein synthesis was stimulated in nonmammary tissues of the body, presumably skeletal muscle.

Background Dietary selenium has the potential to reduce growth of mammary tumors. Increasing the Se content of cows’ milk proteins is a potentially effective means to increase Se intake in humans. We investigate the effects of selenized milk protein on human mammary tumor progression in immunodeficient BALB/c nude mice. Methods Four isonitrogenous diets with selenium levels of 0.16, 0.51, 0.85 and 1.15 ppm were formulated by mixing low- and high-selenium milk casein isolates with a rodent premix. MCF-7 cells were inoculated into the mammary fat pad of female BALB/c nude mice implanted with slow-release 17 β-estradiol pellets. Mice with palpable tumors were randomly assigned to one of the four diets for 10 weeks, during which time weekly tumor caliper measurements were conducted. Individual growth curves were fit with the Gompertz equation. Apoptotic cells and Bcl-2, Bax, and Cyclin D1 protein levels in tumors were determined. Results There was a linear decrease in mean tumor volume at 70 days with increasing Se intake (P < 0.05), where final tumor volume decreased 35% between 0.16 and 1.15 ppm Se. There was a linear decrease in mean predicted tumor volume at 56, 63 and 70 days, and the number of tumors with a final volume above 500 mm3, with increasing Se intake (P < 0.05). This tumor volume effect was associated with a decrease in the proportion of tumors with a maximum growth rate above 0.03 day-1. The predicted maximum volume of tumors (Vmax) and the number of tumors with a large Vmax, were not affected by Se-casein. Final tumor mass, Bcl-2, Bax, and Cyclin D1 protein levels in tumors were not significantly affected by Se-casein. There was a significantly higher number of apoptotic cells in high-Se tumors as compared to low-Se tumors. Conclusions Taken together, these results suggest that turnover of cells in the tumor, but not its nutrient supply, were affected by dairy Se. We have shown that 1.1 ppm dietary Se from selenized casein can effectively reduce tumor progression in an MCF-7 xenograft breast cancer model. These results show promise for selenized milk protein as an effective supplement during chemotherapy.

A simple method of catheterizing an artery which supplies the ipsilateral side of the mammary gland of a high-producing dairy cow is described. The preparation allows infusion of the mammary gland with nutrients or endocrine factors locally, but does not require general anaesthesia or deep surgical dissection. Following a preliminary experiment involving infusion of acetate, the catheters were removed and the cows returned to the milking herd. Key words: Cow, mammary gland, arterial infusion, acetate

SummaryA dynamic mathematical model of blood flow regulation in the mammary glands of a lactating cow was constructed from a principle of local vasodilator release in response to changes in intracellular adenylate charge. An equation was derived to predict uptake of the milk precursors acetate, glucose, amino acids and fatty acids, as affected by mammary blood flow rate. Metabolism of the precursors to milk components and CO2 was simulated with a set of empirically derived equations. Relative rates of ATP production and utilization regulated both the number of perfused capillaries and the conductance of arteriolar segments in the mammary glands. The model simulated local control phenomena of functional and reactive hyperaemia, and simulation of autoregulation under changing arterial pressure suggested a predominance of precapillary sphincter regulation. It was predicted that an increase in blood flow without the mammary capacity to utilize blood metabolites efficiently would be detrimental to milk synthesis. Conversely, increased blood flow through changes in mammary activity resulted in predictions of higher milk production. It was proposed that the equation for uptake,be used in analysis of mammary arteriovenous differences.

The molecular mechanisms by which cellular energy status regulates global protein synthesis in mammary epithelial cells have not been characterized. The objective of this study was to examine the effect of AMP-activated protein kinase (AMPK) activation by 2-deoxyglucose on protein synthesis and the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway in bovine mammary epithelial cells. Phosphorylation of AMPK at Thr172 increased by 1.4-fold within 5 min, and remained elevated throughout a 30-min time course, in response to 2-deoxyglucose. Global rates of protein synthesis declined by 78% of control values. The decline in protein synthesis was associated with repression of mTORC1 signaling, as indicated by reduced phosphorylation of ribosomal protein S6 kinase 1 and eIF4E binding protein-1 (4E-BP1). Phosphorylation of ER-stress marker eIF2α was also increased but only at 30 min of 2-deoxyglucose exposure. 2-Deoxyglucose increased phosphorylation of tuberous sclerosis complex 2 (TSC2) on AMPK consensus sites but did not change the amount of TSC1 bound to TSC2. Activation of AMPK did not result in changes in the amount of raptor bound to mTOR. The inhibitory effects of AMPK activation on mTORC1 signaling were associated with a marked increase in Ser792 phosphorylation on raptor. Collectively, the results suggest that activation of AMPK represses global protein synthesis in mammary epithelial cells through inhibition of mTORC1 signaling.

We used a proteomics-based approach to investigate potential regulatory proteins in the pancreas of domestic cattle (Bos taurus) that were associated with differences in pancreatic α-amylase activity. Two groups of 48 and 45 crossbred steers in years 1 and 2, respectively, were fed a high moisture corn-based diet and were ranked according to their pancreatic α-amylase activity. Steers (n=18) with high, medium, and low α-amylase activity were selected, with 3 for each activity range and 9 for each experimental year, and their proteomic profiles were compared. Pancreatic samples from each animal were fractionated using 2D-HPLC and fractions detected using UV spectrophotometry. Software analysis revealed 119 common protein fractions among the 18 animals, and statistical analysis revealed 10 of these fractions differing (P0.10) in abundance between animals from the high and low pancreatic α-amylase activity groups. Five protein fractions identified after tandem mass spectrometry analysis and database searches were found to match proteins with protein-binding, nucleotide/DNA-binding or enzymatic capabilities. Bioinformatics analysis of these fractions revealed porphobilinogen deaminase, a DNA-binding protein, and a putative S1 peptidase that increased in abundance with increasing α-amylase activity; with a putative ATP/GTP binding protein decreasing in abundance with increasing pancreatic α-amylase activity. Changes in these fractions may represent adaptations of the pancreas in domestic cattle that are associated with differences in α-amylase activity.

Ruminants absorb little glucose and rely on hepatic gluconeogenesis and ketogenesis in the fed state to convert short-chain fatty acids produced during digestion into glucose and ketone bodies, respectively. In contrast to the non-ruminant response, fluxes through gluconeogenic and ketogenic pathways decrease during food restriction. Transcriptional regulation responsible for these unique food restriction responses has not been established. To determine the hepatic transcriptional response of ruminants to an acute drop in dietary nutrient supply, 102 yearling heifers were assigned to either ad libitum feeding or 24 h of food withdrawal in a randomized block design. Liver biopsies were obtained for microarray and quantitative real-time PCR analyses of gene expression. Plasma concentrations of non-esterified fatty acids were higher in food restricted heifers, while levels of β-hydroxybutyrate, triacylglycerol, and glucose were decreased. Despite a decline in substrate supply and a lower hepatic production of glucose, expression of the key gluconeogenic enzymes pyruvate carboxylase, phosphoenolpyruvate carboxykinase and fructose-1,6-bisphosphatase was upregulated as in non-ruminants. Downregulation of cholesterolgenic genes and upregulation of fatty acid oxidative genes were consistent with SREBP-2 and PPARα control, respectively. Ketogenesis from short-chain fatty acids was downregulated, contrary to the non-ruminant response to food restriction. Short-chain fatty acids may exert transcriptional control in the ruminant liver similar to that demonstrated in the large intestine of non-ruminants.

Mechanisms of change in milk composition in response to dietary fat supplementation were examined by measurement of plasma metabolite arteriovenous differences across the udders of cows fed fat. Four primiparous cows fitted with ruminal cannulas were assigned to a factorial arrangement of treatments: two diets and two casein infusion sites in a 4 x 4 Latin square design. Diets were formulated with yellow grease at 0 or 4% of DM. An 8% sodium caseinate solution was infused continuously at 5.04 kg/d into the rumen or abomasum during the last 5 d of each 21-d period. Plasma acetate concentration and mammary uptake were not affected significantly by added dietary fat, but plasma triacylglycerol concentration and uptake were increased, which resulted in milk with a greater fat percentage and a higher proportion of long-chain fatty acids. Reduced lipogenesis from acetate and increased β-hydroxybutyrate uptake spared glucose for lactose synthesis, allowing increased milk yield with reduced mammary blood flow rate. Increased energy supply to mammary tissue, resulting from changes in patterns of plasma metabolites with added dietary fat, was implicated in the reduction of mammary blood flow rates.

SUMMARYA dynamic mathematical model of a closed mammary system in lactating cows was developed to incorporate the setpoint concept of tissue activity, using equations where nutrient supply and absorption are locally regulated so as to maintain a given rate of milk protein yield. The model consists of 12 differential equations, 11 of which are concerned with intracellular biochemical compartments and one describes the volume of tissue actively perfused by blood (AP). The intracellular compartments are: amino acids (AAs), acetate, fatty acids (FAs), β-hydroxybutyrate, glucose-6-phosphate, fructose-6-phosphate, phospho-glyceraldehyde, pyruvate, mitochondrial acetyl-CoA, adenosine triphosphate (ATP) and adenosine diphosphate (ADP). The model simulates mechanisms which are aimed at reproducing and, thereby, explain variations in mammary plasma flow (MPF) observed experimentally. The AP changes according to variations in the metabolic status or in the metabolic requirements of the gland. Should the tissue energy charge (i.e. ATP/ADP ratio) exceed a baseline ratio, then AP decreases and consequently MPF declines. Conversely, when milk protein yield increases, AP increases and MPF rises. In the present model, AA uptake by the mammary gland is inhibited by intracellular AAs. It is also assumed that, when milk protein yield diminishes, the respiratory chain and ATP synthesis become uncoupled and consequently ATP yield is reduced. Model evaluation included behavioural analysis and sensitivity analysis. Behaviour analysis was conducted to test whether the model mechanisms reproduced the scenarios from which the model hypotheses were developed, and took into consideration: an increase in arterial glucose concentration (HIGLC), increases in arterial concentrations of non-esterified FAs, triacylglycerol and β-hydroxybutyrate (HIFAT), a 50% reduction of arterial histidine concentration (LOHIS), and a hyperinsulinaemic euglycaemic clamp (HIINS). Both HIGLC and HIFAT resulted in a decrease in MPF and in milk protein yield; moreover, the scenario HIGLC also produced a notable decrease in the extraction of glucose. The scenario LOHIS resulted in increased MPF and extraction of His from plasma. However these responses were not sufficiently large to prevent a severe reduction of milk protein yield which was accompanied by a reduction in the extraction of other essential AAs. The scenario HIINS resulted in an increase of MPF and of milk protein yield, in the extraction of His and of other essential AAs. Model sensitivity analysis focused on variation of both affinity and inhibition constants of some of the Michaelis–Menten equations. Improvements in model structure and directions for future research suggested by the modelling analysis are discussed.

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 (P0.05) or tended to increase (P0.1) 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.

Using the MAC-T cell line as a model, the effects of insulin-like growth factor (IGF)-1 on the regulation of protein synthesis through the mammalian target of rapamycin complex 1 (mTORC1) signaling in bovine mammary epithelial cells were evaluated. Global rates of protein synthesis increased by 47% within 30 min of IGF-1 treatment. The effect of IGF-1 on protein synthesis was associated with enhanced association of the eukaryotic initiation factor (eIF) 4E with eIF4G and a concomitant reduction of eIF4E association with eIF4E-binding protein-1 (4E-BP1). There was a progressive increase in the phosphorylation state of ribosomal protein S6 kinase-1, a downstream target of mTORC1 in response to IGF-1. In addition, IGF-1 stimulated mTORC1 kinase activity toward 4E-BP1 in vitro. Phosphorylation on Ser473 of Akt was induced by IGF-1 within 5 min and remained elevated throughout a 30-min time course. The effect of IGF-1 on Akt phosphorylation was also concentration dependent. Activation of Akt by IGF-1 led to increased phosphorylation of tuberous sclerosis complex 2 on Thr1426, without any change in its association with tuberous sclerosis complex 1. Phosphorylation of proline-rich Akt substrate of 40-kDa (PRAS40) at Thr246 was stimulated by IGF-1. The amount of PRAS40 associated with mTORC1 decreased in response to IGF-1, and PRAS40 binding to mTORC1 was inversely related to its phosphorylation level. Overall, these results suggest that activation of the PI3K-Akt pathway by IGF-1 stimulated global protein synthesis in bovine mammary epithelial cells through changes in the phosphorylation and association state of components of the mTORC1 signaling pathway.

International audience; Although amino acids (AA) synthesized by enteric microbiota in the upper gut of nonruminants can be absorbed, they do not necessarily make a net contribution to the host's AA supply. That depends on whether protein or nonprotein nitrogen sources are used for microbial protein production. We determined the contributions of urea, endogenous protein (EP), and dietary protein (DP) to microbial valine (M.VAL) at the distal ileum of growing pigs, based on isotope dilutions after a 4-d continuous infusion of l-[1-(13)C]valine to label EP and of [(15)N(15)N]urea. Eight barrows were assigned to either a cornstarch and soybean meal-based diet with or without 12% added fermentable fiber from pectin. Dietary pectin did not affect (P > 0.10) the contributions of the endogenous and DP to M.VAL. More than 92% of valine in microbial protein in the upper gut was derived from preformed AA from endogenous and DP, suggesting that de novo synthesis makes only a small contribution to microbial AA.

Perturbation of the amino acid status of lactating dairy cows was undertaken to characterize the mechanism of dietary fat-induced milk protein depression. Four first lactation heifers fitted with rumen cannulas were assigned to a factorial arrangement of treatments: two diets and two casein infusion sites within a 4 x 4 Latin square design. Diets were formulated with yellow grease at 0 or 4% of DM. An 8% sodium caseinate solution was infused continuously at 5.04 kg/d (SE = .05) into the rumen or abomasum during the last 5 d of each 21-d period. Dry matter, N, energy, and NDF digestibilities were reduced by dietary fat treatment, but intakes of digestible DM, N, and energy were not affected. Yellow grease supplementation improved milk yield 14% and milk fat content 7%, whereas lactose, CP, and casein N percentages in milk were reduced. Abomasal sodium caseinate infusions had no effect on milk yield or composition other than to increase protein and casein N concentrations. Dietary fat-induced milk protein depression remained evident during abomasal casein treatments, indicating a mechanism independent of amino acid status of the cow. Improved intestinal availability of amino acids partially reversed detrimental effects of dietary fat on milk protein fractions.

An experiment was conducted to test the hypothesis that a sufficient proportion of histidine (His) included in the drinking water of lactating cows bypasses the rumen to have an effect on milk synthesis. Eight dairy cows (45 +/- 15 d in milk) were given either 0 or 2.5 g/L of His in the drinking water in a crossover design of two 7-d periods. Cows were offered a corn and alfalfa silage-based total mixed ration for ad libitum intake. Water was provided ad libitum to each cow in an individual automatic drinking vessel with a flow meter attached. Water intake tended to increase from 85.1 to 92.1 L/d when His was added. Concentrations of His in plasma samples collected on the last day of each period tended to increase from 14.6 to 21.6 muM, corresponding to an estimated 0.4% bypass of the imbibed histidine. Other amino acid concentrations in plasma were not affected by His supplementation. Milk yield increased by 1.7 L/d with His treatment, lactose yield increased by 90 g/d, and there were tendencies for protein yield to increase, fat percentage to decrease, and protein to fat ratio to increase. An improvement in postruminal histidine flow can influence milk production and composition but the proportion of imbibed water that bypasses the rumen will have to be increased to take advantage of drinking water as a vehicle to transfer His postruminally.

Low concentrations of the essential amino acid histidine in circulation have been shown to increase mammary blood flow and it has been suggested that this effect is mediated by histamine. The hypotheses tested in this experiment were that interstitial histamine concentrations in the mammary gland are related to arterial His concentrations and that mammary blood flow is reduced by extracellular histamine via H(1) receptors. The hypotheses were tested by infusing saline or chlorpheniramine, a blocker of the H(1) histamine receptor, into the arterial supply of the mammary glands of lactating cows infused with 44 g/h of amino acid mixtures with or without His for 10 h. Infusates were administered in a 2 x 2 factorial arrangement within a 4 x 4 Latin square to 4 multiparous Holstein cows in mid lactation. Exclusion of His from the infusate decreased protein content in milk from the infused udder half from 3.98 to 3.77%, and increased arterial alpha-aminonitrogen concentration from 3.2 to 3.4 mM. Neither the decreased arterial His concentration nor the H(1) blocker affected plasma flow to the infused udder half. We conclude that histamine is not involved in the regulation of mammary blood flow. The H(1) blocker decreased milk production in the infused udder half from 4.6 to 3.5 kg without affecting protein, fat, and lactose percentages, suggesting an inhibition of milk ejection. Cows on chlorpheniramine ate less feed during the infusion than saline-infused cows, which resulted in lower arterial concentrations and mammary uptakes of acetate. The efficiency of plasma triacylglycerol uptake across the mammary glands was decreased by chlorpheniramine but net uptake of long-chain fatty acids was not affected. The mechanism by which an amino acid deficiency influences mammary blood flow does not involve histamine signaling through the H(1) receptor and remains unidentified.

Rates of secretion of components into milk are a function of precursor concentrations and parameters that describe expression of the milk synthetic enzymes and their sensitivity to precursor concentrations. To establish the enzymatic sensitivities of milk fat yield and mammary acetate utilization to circulating acetate concentration, lactating cows were infused for 10 h with 0 or 40 g of acetate/h in an external iliac artery supplying one udder half. In addition, to investigate the possibility that energy supply influences the milk protein response to an elevated amino acid (AA) concentration, 2 different AA profiles were infused with and without acetate. Six cows, fed a total mixed ration of 21% crude protein ad libitum, were infused with AA at 0 g/h, 30 g/h in the profile of rumen microbes, or 30 g/h in the profile of milk proteins, in a 3 x 2 factorial arrangement with the 2 acetate treatments of 0 and 40 g/h, all in a 6 x 6 Latin square. Amino acid infusion caused a 60% increase, on average, in plasma concentration of AA entering the infused udder half. From the microbial AA profile, 49% of infused AA were taken up by the udder half, 42% of which occurred during the first pass. From the milk AA profile, 44% of infused AA were taken up by the udder half, 50% of which occurred during the first pass. There was an 8% increase in yield of milk protein with AA infusion, representing 7% capture, but no effect of the infused profile. Acetate infusion caused a decrease in the yields of milk protein and lactose when AA were infused, but not when AA were absent. Milk fat yields were not affected, although acetate concentrations in plasma entering the infused udder half increased by 123% and mammary uptakes increased by 128%. Mammary uptakes of long-chain fatty acids and beta-hydroxybutyrate were not affected by acetate infusion, whereas glucose uptakes tended to increase. It was suggested that excess acetate may have been sequestered in adipose tissue in the udder. Yields of both protein and fat in milk showed a low sensitivity to the concentration of their precursors in circulation. It was concluded that the Km in Michaelis-Menten-type equations describing milk synthesis should be assigned a low value, and that the Vmax is regulated to bring about changes in milk yield and composition.

To test if control of mRNA translation is involved in the increase in protein synthesis by mammary glands during lactation, cellular contents and phosphorylation states of translation factors and their upstream regulators were measured in mammary parenchyma from 12 nonpregnant dairy cows. For a 42-d period, 6 cows in late lactation continued to be milked (L) and 6 at the same stage of lactation were dried off (NL). All cows were then slaughtered and mammary glands and tissue samples obtained. Alveoli and lobules tended to be larger in L cows. Mammary parenchymal mass, cell number, cell size, and RNA, DNA, and protein contents were greater in L cows. Increases (3.1- and 1.8-fold) in the abundance of active, phosphorylated ribosomal protein S6 and its kinase, S6K1, respectively, in L vs. NL parenchyma indicated an ability to sustain greater rates of synthesis of translational machinery, which was also evident in the 102% increase in parenchymal RNA:DNA between the 2 groups. Cellular abundances of the main eukaryotic translation initiation factors (eIF), eIF2 and eIF4E, were 2.6- and 3-fold greater, respectively, in L cows. That these differences were greater than the 102% greater RNA:DNA in L mammary parenchyma suggests an elevated translational efficiency in L glands. Abundance of phosphorylated rpS6 was not different between mammary parenchyma and liver, whereas eIF2alpha was 50% greater in mammary tissue. In semimembranosus muscle, abundances of phosphorylated rpS6 and eIF2alpha were 3 to 4 times lower than in mammary parenchyma. In both L and NL mammary glands, 11% of eIF2alpha was in the inhibitory, phosphorylated form and 48 to 60% of eIF4E was complexed with its binding protein, 4EBP1. It is concluded that up-regulation of initiation of mRNA translation occurs in the fully differentiated milk secretory cell and that, where crucial initiation factors are not present in a maximally active form, the initiation rate might be flexible in response to external stimuli.

Glucose transport by isolated bovine mammary epithelial cells involves translocation across the cell membrane into a compartment that exchanges slowly with the bulk cytosol. The significance to glucose metabolism of this compartmentalization was examined by generation, modeling, and analysis of transport and metabolism data. Net uptake of 5m M 3-O-methyl-d-glucose by isolated bovine mammary epithelial cells was measured at 37°C. Time-course curves were better fitted by a double exponential equation than a single exponential equation and were subjected to compartmental analysis to obtain glucose transport model parameters. Lactose synthesis and glucose oxidation rates and cellular concentrations of intermediary metabolites, glucose-6-phosphate and glucose-1-phosphate, were measured at varied media glucose concentrations. A model that integrates both glucose transport and metabolism under-predicted the rates of lactose synthesis and glucose oxidation by a factor of 3. To account for the observed glucose use rates, glucose must be available for phosphorylation once translocated across the cell membrane (intermediate compartmentalization of translocated glucose does not exclude access to hexokinase). Metabolic control analysis indicated that, at physiological glucose concentrations, phosphorylation by hexokinase exerts 80% of the control of glucose metabolism to lactose and CO 2 , and transport exerts the remaining 20%.

Four multiparous ruminally fistulated Holstein dairy cows were used in an 8-wk experiment utilizing a repeated measures block design to determine the effects of subacute ruminal acidosis (SARA) on supplemented water intake. Animals were subjected to SARA, which was induced by replacing 25% of the ad libitum intake of the total mixed ration (dry matter basis) with 50:50 wheat:barley pellets utilizing a grain challenge model. Cows had free choice from 2 water bowls. One bowl contained water with sodium bicarbonate (SB) supplemented at 2.5 g/L. The other bowl contained unsupplemented water. Ruminal pH was monitored continuously during the trial using indwelling pH probes. The induction of SARA reduced daily mean ruminal pH and increased the duration when ruminal pH was below 6. The total mixed ration intake by the cows decreased during the SARA periods. The overall preference for SB-supplemented water did not change, as the preference ratio was similar during the control and SARA periods. During the period of greatest ruminal pH depression, total water intake was higher during the SARA periods than during the control periods. During SARA, there was no difference in the preference of a SB water source to unsupplemented water. During the period of day with the most severe ruminal pH depression, the lactating dairy cows subjected to SARA increased their total water intake.

Glucose transport kinetics were quantified in isolated bovine mammary epithelial cells using 3- O-methyl-d-glucose. Isolated cells retained satisfactory viability and glucose uptake activity, which was inhibited by cytochalasin B, phloretin, HgCl2, and low temperature. Initial rates of entry were measured over a 15-s interval at 37°C under zero- trans, equilibrium-exchange, high- cis, and high- trans concentrations of 3- O-methyl-d-glucose between 0 and 20 mM. The combined set of rate measurements from all experimental conditions was fit to the fixed-site carrier model by nonlinear regression to estimate parameters of transport. For the regression between predicted and observed initial rates, r2was 0.97. Forward Vmaxwas estimated at 18.2 nmol·min-1·mg protein-1, and the Michaelis constant was 8.29 mM. The cooperativity parameter was 1.63, trans-stimulation was 2.13-fold, and asymmetry was 2.06-fold. On the basis of the kinetic parameters, variations in intracellular glucose concentrations are not responsible for the range of glucose uptakes by bovine mammary glands observed in vivo.

Protein is a very important component of cow’s milk, both nutritionally for the consumer and economically for the dairy farmer. In order to manage milk protein production at the farm level, it would be advantageous to have an understanding of its biological regulation that was adequate to predict daily yield. Most of our information regarding regulation of milk protein secretion is derived from arteriovenous difference methodology in which the balance of milk precursors and products is measured across the udder of lactating cows. The purpose of the models presented here is to study the connection between arterial concentrations of milk precursors and the composition of milk produced by bovine mammary glands. More specifically, the models should predict the secretion rate of milk protein by dairy cows, the percentage of milk volume that is protein (this is a factor in many milk pricing schemes), and the arteriovenous differences of milk precursors across the mammary glands (the intermediate pieces of information upon which the knowledge base has been built).

Amino acids (AA) and/or somatotropin (ST) were infused into a jugular vein of lactating cows receiving atropine to determine the effect on milk protein secretion. Atropine decreased milk protein yield by 35%. Plasma alpha-amino nitrogen, which was decreased by 31% due to atropine, was restored to control levels upon AA infusion. Plasma ST concentration was not affected by atropine and, upon ST infusion, increased by 236% in treated animals. Amino acids, ST, or the combination of the two were unable to return milk protein to control levels in atropine-treated animals. Plasma insulin concentration decreased by 37% in atropine-treated animals and was not significantly different for cows receiving atropine as well as ST, AA, or both ST and AA. Similarly, glucagon decreased by 36% in atropine-treated animals but was partially restored in cows receiving AA. Overall, the insulin-to-glucagon ratio was not significantly affected by treatment. Plasma glucose concentration was not affected by treatment. These data lend support to the importance of the insulin-to-glucagon ratio in terms of whole-body metabolism, with the exception of the mammary glands, which are glucagon-insensitive and, therefore, sensitive to the observed decrease in circulating insulin concentration.

Supplemental fat fed to dairy cows affects the fat composition of milk by reducing the yield of mammary synthesized fatty acids. The effect has been attributed to a potential allosteric inhibition of acetyl coenzyme-A, a key enzyme in fatty acid synthesis. In vitro experiments have demonstrated an inhibition of fatty acid synthesis when long-chain fatty acids are added to incubations. However, in vitro inhibition can result from a nonspecific detergent effect arising from an inherent physical property of fatty acids. An allosteric role for palmitic acid has not been tested in bovine mammary tissue. The objective of this experiment was to test the hypothesis that palmitic acid is an allosteric inhibitor of fatty acid synthesis in mammary tissue. We tested for a detergent effect by including a synthetic detergent, sodium dodecyl sulfate, under identical incubation conditions. A subcellular supernatant fraction of mammary tissue was used for incubations in the present experiment. The incubation system produced free fatty acids in a linear fashion for time and protein content. Results indicated that fatty acid synthesis was affected by the addition of palmitic acid to the incubations but not by caprylic acid, a short-chain fatty acid. Sodium dodecyl sulfate did not affect fatty acid synthesis at the concentrations used. The results of the present experiment indicate that palmitic acid inhibited fatty acid synthesis, and the effect was not the result of a detergent effect.

Concentrations of glucose in the external iliac artery feeding one udder half of 14 midlactation Holstein cows were increased by infusion to test the following three hypotheses of mammary function: 1) that mammary glands control their blood supply to maintain intracellular energy balance, 2) that milk precursors are taken out of capillary blood according to mass action kinetics, and 3) that the rate of milk component synthesis is dependent on its precursor's uptake from blood. The first seven cows received 20 g/h glucose during 10 h of infusion. Arterial concentrations of glucose were locally increased by only 10%, and the iliac plasma flow was not affected by glucose infusion, so the next seven cows were given 90 g/h glucose. Quantitative predictions resulting from the hypotheses were that arterial plasma flow would decrease by 32% with 90 g/h glucose infusion, glucose uptakes would increase and acetate, fatty acid, and amino acid uptakes decrease, and milk protein and fat yields and percentages would decrease. Iliac plasma flow decreased 16%, half of what was predicted, which suggests that other regulatory processes besides blood flow control took part in the response. Acetate and fatty acid uptakes by the mammary glands were reduced as predicted because of the lower blood flow, but an unexpected depression in extraction of plasma triacylglycerol also contributed to the reduced fatty acid uptake. Milk fat and protein yields were not affected by the exogenous glucose, falsifying the third hypothesis that milk component secretion is a function of uptake of its precursor. Milk fat and protein percentages declined with glucose infusion because of increased lactose synthesis and secretion of water into milk.

Changes in mammary AA utilization associated with dietary fat-induced milk protein depression were assessed in cows with disturbed AA status. Four first lactation cows fitted with rumen cannulas were assigned to a factorial arrangement of treatments: two diets and two casein infusion sites within a 4 x 4 Latin square design. Diets were formulated with yellow grease at 0 or 4% of DM. An 8% sodium caseinate solution was infused continuously at 5.04 kg/d into the rumen or abomasum during the last 5 d of each 21-d period. Arterial AA concentrations were reduced by dietary fat treatment and increased by abomasal sodium caseinate infusion. Mammary arteriovenous differences of essential AA tended to increase with both abomasal casein infusion and dietary fat treatments. With ruminal casein infusion treatments, yellow grease supplementation increased the percentage of extraction of blood essential AA into mammary tissue. Mammary blood flow rate dropped 7% on high fat treatments, preventing an increase in uptakes of critical AA to accompany the improved efficiency of milk synthesis, which was evident from a significantly reduced ratio of mammary blood flow to milk volume, resulting in depressed milk protein content.

In digesting and absorbing dietary nutrients, the gastrointestinal tract consumes approximately 20% of all incoming energy. A substantial proportion of this consumption is due to the rapid turnover of cellular protein, which permits abrupt changes in gut size to occur, matching capacity with delivery. If it is size of the alimentary tract that constrains nutrient uptake, greater than 20% allocation of ME intake above maintenance to the gut would improve the growth rate of a young animal but the efficiency of ME utilization for growth would deteriorate. Less than 15% allocation in birds seems injurious to both growth rate and efficiency of growth. Nutrient transport capacity of the intestine may be modulated independent of size; in the case of glucose, an up- or down-regulation of the number of brush-border glucose transporters matches absorptive capacity with delivery. The maximum uptake capacity of a small intestine for glucose at any moment in time is a function of its length, the flow rate of digesta, and the distributed-in-space kinetic parameters of transport (e.g., Vmax and Km). An example maximum uptake capacity for glucose in sheep is calculated at 2,112 g/d, assuming continuous digesta flow. Intermittency of flow reduces the uptake capacity to a functional level of 295 g/d, demonstrating a constraining influence of the periodicity of the migrating myoelectric complex. Growth regulation by stimulatory and inhibitory mitotic signals is presented as a candidate for an energy-independent determinant of the upper limit to functional maximum uptake capacity of the small intestine. Both size and functional capacity of the intestine must be considered in assessing the impact this tissue may have on the rest of the animal.

In this review, we discuss the mechanismsof responses of various tissues of the lactating dairy cow, particularly the mammary glands, to perturbations in supply of single amino acids that result in observed milk protein yields. Additions of methionine, lysine, histidine or leucine to the absorptive supply cause arterial concentrations of these amino acids to increase, mammary extractions to drop and mammary blood flow to decrease. Single subtractions of essential amino acids have the opposite effect. Changes in mammary blood flow that have been recorded can be explained as attempts by the mammary glands to restore intracellular ATP balance in the face of altered concentrations of energy metabolites in the general circulation. In a quantitative sense, milk protein yield is relatively insensitive to fluctuations in arterial amino acid concentrations but can be stimulated by any one of a number of amino acids. In this context, it is suggested that the designation of a limiting amino acid is not appropriate to the purpose of predicting milk protein yield. Rather, milk protein synthesis appears to operate at a predetermined rate set by external communications of milk withdrawal rate, physiological state and overall nutritional status. Utilization of amino acids (AA) by splanchnic and peripheral tissues, in coordination with the mammary setpoint, offsets imperfections in the dietary AA supply. How strongly an individual AA influences the mammary setpoint, arterial concentrations of energy metabolites, and mammary AA transport capacity will determine the magnitude of the milk protein yield response when its absorptive supply is changed. Key words: Dairy cows, milk protein, amino acid