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Essential amino acid profile of supplemental metabolizable protein affects mammary gland metabolism and whole-body glucose kinetics in dairy cattle

Open AccessPublished:July 18, 2022DOI:https://doi.org/10.3168/jds.2021-21576

      ABSTRACT

      This study investigated mammary gland metabolism and whole-body (WB) rate of appearance (Ra) of glucose in dairy cattle in response to a constant supplemental level of metabolizable protein (MP) composed of different essential AA (EAA) profiles. Five multiparous rumen-fistulated Holstein-Friesian dairy cows (2.8 ± 0.4 lactations; 81 ± 11 d in milk; mean ± standard deviation) were abomasally infused according to a 5 × 5 Latin square design with saline (SAL) or 562 g/d of EAA delivered in different profiles where individual AA content corresponded to their relative content in casein. The profiles consisted of (1) a complete EAA mixture (EAAC), (2) Ile, Leu, and Val (ILV), (3) His, Ile, Leu, Met, Phe, Trp, Val (GR1+ILV), and (4) Arg, His, Lys, Met, Phe, Thr, Trp (GR1+ALT). A total mixed ration (58% corn silage, 16% alfalfa hay, and 26% concentrate on a dry matter basis) was formulated to meet 100 and 83% of net energy and MP requirements, respectively, and was fed at 90% of ad libitum intake on an individual cow basis. Each experimental period consisted of 5 d of continuous abomasal infusion followed by 2 d of no infusion. Arterial and venous blood samples were collected on d 4 of each period for determination of mammary gland AA and glucose metabolism. On d 5 of each period, D-[U-13C]glucose (13 mmol priming dose; continuous 3.5 mmol/h for 520 min) was infused into a jugular vein and arterial blood samples were collected before and during infusion to determine WB Ra of glucose. Milk protein yield did not differ between EAAC, GR1+ILV, and GR1+ALT, or between SAL and ILV, and increased over SAL and ILV with EAAC and GR1+ILV. Mammary plasma flow increased with ILV infusion compared with EAAC and GR1+ILV. Infusion of EAAC tended to increase mammary gland net uptake of total EAA and decreased the mammary uptake to milk protein output ratio (U:O) of non-EAA compared with SAL. Infusion of ILV increased mammary net uptake and U:O of Ile, Leu, and Val markedly over all treatments. The U:O of total Ile, Leu, and Val increased numerically (25%) with GR1+ILV infusion compared with EAAC, and the U:O of total Arg, Lys, and Thr tended to decrease, primarily from decreased U:O of Lys. During GR1+ALT infusion, U:O of total Arg, Lys, and Thr was greater than that during EAAC infusion, whereas U:O of Ile, Leu, and Val did not differ from EAAC. Glucose WB Ra increased 16% with GR1+ALT over SAL, and increased numerically 8 and 12% over SAL with EAAC and GR1+ILV, respectively. The average proportion of lactose yield relative to glucose WB Ra did not differ across treatments and averaged 0.53. On average, 28% of milk galactose arose from nonglucose precursors, regardless of treatment. In conclusion, intramammary catabolism of group 2 AA increased to support milk component synthesis when the EAA profile of MP was incomplete with respect to casein. Further, WB and mammary gland glucose metabolism was flexible in support of milk component synthesis, regardless of absorptive EAA profile.

      Key words

      INTRODUCTION

      Mammary gland uptake of AA for milk protein synthesis plays a key role in the efficiency of transfer of dietary N into milk N (
      • Cant J.P.
      • Berthiaume R.
      • Lapierre H.
      • Luimes P.H.
      • McBride B.W.
      • Pacheco D.
      Responses of the bovine mammary glands to absorptive supply of single amino acids.
      ;
      • Haque M.N.
      • Guinard-Flament J.
      • Lamberton P.
      • Mustière C.
      • Lemosquet S.
      Changes in mammary metabolism in response to the provision of an ideal amino acid profile at 2 levels of metabolizable protein supply in dairy cows: Consequences on efficiency.
      ). In order for mammary EAA uptake to be increased, accompanying responses in milk protein synthesis, tissue protein accretion, or intramammary AA catabolism must occur (
      • Cant J.P.
      • Kim J.J.M.
      • Cieslar S.R.L.
      • Doelman J.
      Symposium review: Amino acid uptake by the mammary glands: Where does the control lie?.
      ). Of the EAA, mammary gland net transfer of group 1 AA (His, Met, Phe+Tyr, and Trp) into milk canonically occurs in a 1:1 ratio with their uptake from arterial circulation. A defining characteristic of group 2 AA (Arg, Ile, Leu, Lys, Thr, and Val) is their excess mammary net uptake relative to their output in milk protein (
      • Mepham T.B.
      Amino acid utilization by lactating mammary gland.
      ;
      • Lapierre H.
      • Lobley G.E.
      • Doepel L.
      • Raggio G.
      • Rulquin H.
      • Lemosquet S.
      Triennial Lactation Symposium: Mammary metabolism of amino acids in dairy cows.
      ). Intramammary metabolism of the excess group 2 AA, particularly the branched-chain AA (Ile, Leu, and Val), Arg, and Lys, provides substantial N and carbon for de novo NEAA synthesis, and for glycolytic and tricarboxylic acid cycle intermediates (
      • Bequette B.J.
      • Backwell F.R.C.
      • MacRae J.C.
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      Effect of intravenous amino acid infusion on leucine oxidation across the mammary gland of the lactating goat.
      ;
      • Mabjeesh S.J.
      • Kyle C.E.
      • MacRae J.C.
      • Bequette B.J.
      Lysine metabolism by the mammary gland of lactating goats at two stages of lactation.
      ;
      • Lapierre H.
      • Doepel L.
      • Milne E.
      • Lobley G.E.
      Responses in mammary and splanchnic metabolism to altered lysine supply in dairy cows.
      ). The mammary gland may adapt to deficiencies of single EAA by altering the rate of blood flow to the tissue (
      • Bequette B.J.
      • Backwell F.R.C.
      • MacRae J.C.
      • Lobley G.E.
      • Crompton L.A.
      • Metcalf J.A.
      • Sutton J.D.
      Effect of intravenous amino acid infusion on leucine oxidation across the mammary gland of the lactating goat.
      ,
      • Bequette B.J.
      • Hanigan M.D.
      • Calder A.G.
      • Reynolds C.K.
      • Lobley G.E.
      • MacRae J.C.
      Amino acid exchange by the mammary gland of lactating goats when histidine limits milk production.
      ;
      • Doepel L.
      • Hewage I.I.
      • Lapierre H.
      Milk protein yield and mammary metabolism are affected by phenylalanine deficiency but not by threonine or tryptophan deficiency.
      ), cellular AA transporter activity (
      • Baumrucker C.R.
      Amino acid transport systems in bovine mammary tissue.
      ;
      • Maas J.A.
      • France J.
      • Dijkstra J.
      • Bannink A.
      • McBride B.W.
      Application of a mechanistic model to study competitive inhibition of amino acid uptake by the lactating bovine mammary gland.
      ;
      • Bequette B.J.
      • Hanigan M.D.
      • Calder A.G.
      • Reynolds C.K.
      • Lobley G.E.
      • MacRae J.C.
      Amino acid exchange by the mammary gland of lactating goats when histidine limits milk production.
      ), the level of intramammary AA catabolism (
      • Lapierre H.
      • Doepel L.
      • Milne E.
      • Lobley G.E.
      Responses in mammary and splanchnic metabolism to altered lysine supply in dairy cows.
      ), the rate of protein synthetic activities (
      • Doelman J.
      • Kim J.J.M.
      • Carson M.
      • Metcalf J.A.
      • Cant J.P.
      Branched-chain amino acid and lysine deficiencies exert different effects on mammary translational regulation.
      ,
      • Doelman J.
      • Curtis R.V.
      • Carson M.
      • Kim J.J.M.
      • Metcalf J.A.
      • Cant J.P.
      Essential amino acid infusions stimulate mammary expression of eukaryotic initiation factor 2Bε but milk protein yield is not increased during an imbalance.
      ), or through a combination of these, in an effort to maintain milk protein synthesis.
      The liver removes AA in coordination with maintenance of whole-body (WB) AA homeostasis, substrate supply for gluconeogenesis, AA requirements for constituent and export proteins, and AA requirements of peripheral tissues (
      • Omphalius C.
      • Lemosquet S.
      • Ouellet D.R.
      • Bahloul L.
      • Lapierre H.
      Postruminal infusions of amino acids or glucose affect metabolisms of splanchnic, mammary, and other peripheral tissues and drive amino acid use in dairy cows.
      ). Of the EAA, His, Met, and Phe are susceptible to hepatic sequestration that usually increases with increasing supply, whereas liver extraction of Ile, Leu, and Val is negligible across a range of MP supplies (
      • Raggio G.
      • Pacheco D.
      • Berthiaume R.
      • Lobley G.E.
      • Pellerin D.
      • Allard G.
      • Dubreuil P.
      • Lapierre H.
      Effect of level of metabolizable protein on splanchnic flux of amino acids in lactating dairy cows.
      ;
      • Omphalius C.
      • Lemosquet S.
      • Ouellet D.R.
      • Bahloul L.
      • Lapierre H.
      Postruminal infusions of amino acids or glucose affect metabolisms of splanchnic, mammary, and other peripheral tissues and drive amino acid use in dairy cows.
      ). Propionate is recognized as the major glucose precursor in fed lactating ruminants, but absorbed AA (except Leu and Lys) can make net contributions to hepatic gluconeogenesis, ranging from 2 to 40% (
      • Danfær A.
      • Tetens V.
      • Agergaard N.
      Review and an experimental study on the physiological and quantitative aspects of gluconeogenesis in lactating ruminants.
      ;
      • Huntington G.B.
      • Harmon D.L.
      • Richards C.J.
      Sites, rates, and limits of starch digestion and glucose metabolism in growing cattle.
      ). Whole-body glucose flux generally increases in response to increased protein supply (
      • Clark J.H.
      • Spires H.R.
      • Derrig R.G.
      • Bennink M.R.
      Milk production, nitrogen utilization and glucose synthesis in lactating cows infused postruminally with sodium caseinate and glucose.
      ;
      • Galindo C.E.
      • Ouellet D.R.
      • Pellerin D.
      • Lemosquet S.
      • Ortigues-Marty I.
      • Lapierre H.
      Effect of amino acid or casein supply on whole-body, splanchnic, and mammary glucose kinetics in lactating dairy cows.
      ), but the magnitude of contribution of total AA to glucose production varies, mainly due to physiological and nutrient status (i.e., lactation stage, and the balance in energy and protein supply relative to animal requirements;
      • Danfær A.
      • Tetens V.
      • Agergaard N.
      Review and an experimental study on the physiological and quantitative aspects of gluconeogenesis in lactating ruminants.
      ).
      Apparent differences in AA metabolism by splanchnic and peripheral tissues suggests that the AA profile of MP is important when aiming to improve the transfer of dietary N into milk N. In primary mammary epithelial cells,
      • Yoder P.S.
      • Ruiz-Cortes T.
      • Castro J.J.
      • Hanigan M.D.
      Effects of varying extracellular amino acid profile on intracellular free amino acid concentrations and cell signaling in primary mammary epithelial cells.
      described the capability of several EAA profiles to stimulate protein translation pathways in mammary cells, suggesting the search for a single stimulating profile is moot. Previous studies have evaluated the effects of EAA deficiencies in duodenal supply on milk protein synthesis, where single AA or groups of AA were subtracted from postruminal infusions of complete AA profiles (
      • Weekes T.L.
      • Luimes P.H.
      • Cant J.P.
      Responses to amino acid imbalances and deficiencies in lactating dairy cows.
      ;
      • Doelman J.
      • Kim J.J.M.
      • Carson M.
      • Metcalf J.A.
      • Cant J.P.
      Branched-chain amino acid and lysine deficiencies exert different effects on mammary translational regulation.
      ,
      • Doelman J.
      • Curtis R.V.
      • Carson M.
      • Kim J.J.M.
      • Metcalf J.A.
      • Cant J.P.
      Essential amino acid infusions stimulate mammary expression of eukaryotic initiation factor 2Bε but milk protein yield is not increased during an imbalance.
      ), but fewer have examined the effects on mammary gland metabolism (
      • Doepel L.
      • Lapierre H.
      Deletion of arginine from an abomasal infusion of amino acids does not decrease milk protein yield in Holstein cows.
      ;
      • Haque M.N.
      • Guinard-Flament J.
      • Lamberton P.
      • Mustière C.
      • Lemosquet S.
      Changes in mammary metabolism in response to the provision of an ideal amino acid profile at 2 levels of metabolizable protein supply in dairy cows: Consequences on efficiency.
      ;
      • Doepel L.
      • Hewage I.I.
      • Lapierre H.
      Milk protein yield and mammary metabolism are affected by phenylalanine deficiency but not by threonine or tryptophan deficiency.
      ) or WB rate of appearance (Ra) of glucose (
      • Lemosquet S.
      • Delamaire E.
      • Lapierre H.
      • Blum J.W.
      • Peyraud J.L.
      Effects of glucose, propionic acid, and nonessential amino acids on glucose metabolism and milk yield in Holstein dairy cows.
      ). Therefore, the first objective of the current experiment was to examine mammary gland responses to incomplete EAA profiles. In this experiment, dairy cows postruminally supplemented with 562 g of EAA/d in profiles where Arg, Lys, and Thr, or Ile, Leu, and Val were absent produced the same level of total milk, protein, fat, and lactose as compared with infusion of a complete EAA profile at the same dose (
      • Nichols K.
      • Bannink A.
      • Dijkstra J.
      Energy and nitrogen balance of dairy cattle as affected by provision of different essential amino acid profiles at the same metabolizable protein supply.
      ). Infusion of only Ile, Leu, and Val resulted in lower milk protein yield compared with the complete EAA profile, but supported the same level of milk protein production as the negative control (saline), despite lower feed intake. Based on the similar milk protein yield produced with supplementation of a complete EAA profile and those lacking Arg, Lys, and Thr or Ile, Leu, and Val, we hypothesized that intramammary catabolism of those EAA would decrease when they were absent from the infusion, and would increase when they were present. We expected intramammary catabolism of the branched-chain AA to increase with infusion of only Ile, Leu, and Val. A second objective of this experiment was to determine if the EAA profile of supplemented MP affected WB Ra of glucose. We hypothesized that differences in affinity of certain AA groups for hepatic metabolism may affect WB Ra of glucose during infusion of incomplete EAA profiles.

      MATERIALS AND METHODS

      Milking, Feeding, and Treatment Infusions

      The experimental procedures were conducted from August to October 2017 at the animal research facilities of Wageningen University & Research (Wageningen, the Netherlands) under the Dutch Law on Animal Experiments in accordance with European Union Directive 2010/63, and were approved by the Central Committee of Animal Experiments (The Hague, the Netherlands). The experimental design, animal housing, ration composition and preparation, and feed chemical analyses have been described in detail by
      • Nichols K.
      • Bannink A.
      • Dijkstra J.
      Energy and nitrogen balance of dairy cattle as affected by provision of different essential amino acid profiles at the same metabolizable protein supply.
      . Briefly, the effects of EAA profiles within a constant supplemented MP level were tested using 5 rumen-fistulated, Holstein-Friesian dairy cows (2.8 ± 0.4 lactations; 81 ± 11 DIM) randomly assigned to a 5 × 5 Latin square design, in which each experimental period consisted of 5 d of continuous abomasal infusion followed by 2 d of no infusion (wash-out period). Cows were housed individually in identical climate respiration chambers [CRC; described in detail by
      • van Gastelen S.
      • Antunes-Fernandes E.C.
      • Hettinga K.A.
      • Klop G.
      • Alferink S.J.J.
      • Hendriks W.H.
      • Dijkstra J.
      Enteric methane production, rumen volatile fatty acid concentrations, and milk fatty acid composition in lactating Holstein-Friesian cows fed grass silage- or corn silage-based diets.
      ], and were allowed 5 d of adaptation to the CRC environment (after an initial 14-d diet adaptation in tiestalls) before the first experimental period began. Cows were fed a TMR (13% CP on DM basis) consisting of 58% corn silage, 16% alfalfa hay, and 26% concentrate on a DM basis which was formulated to meet 100 and 83% of NEL and MP requirements (
      • CVB (Centraal Veevoederbureau)
      CVB Table Ruminants 2008, series nr. 43.
      ), respectively, for cows consuming 21 kg of DM/d and producing 33 kg/d of milk containing 41 g/kg fat and 34 g/kg protein. Daily feed intake for individual cows was restricted to 90% of individual daily ad libitum intake determined during the final 5 d of the tiestall diet adaptation before cows entered the CRC. Fresh feed was allocated twice daily during the entire experiment, with the exception of a 58-h window over d 3 to 5 of each period (from 0530 h on d 3 until 1530 h on d 5), where an automated feeding system dispensed equal portions of feed every 2 h to promote metabolic steady-state conditions in preparation for the blood sampling and isotope infusion protocols described below. Cows had individual and free access to drinking water throughout the entire experiment. Cows were milked twice daily at 0530 and 1530 h. Milk was collected, weighed, and sampled at each milking. Samples were stored at 4°C and analyzed within 4 d for protein, fat, lactose, and urea by mid-infrared spectroscopy (ISO 9622;
      • ISO (International Organization for Standardization)
      Milk and liquid milk products. Guidelines for the application of mid-infrared spectrometry. ISO Standard 9622.
      ).
      Infusion lines were placed in the abomasum via the rumen cannula 2 d before the first experimental period and were checked daily for patency and position. Abomasal infusion treatments were 0.9% saline (SAL) and 4 different AA profiles (562 g/d of anhydrous AA; Table 1) consisting of (1) a complete EAA mixture (EAAC), (2) Ile, Leu, and Val (ILV), (3) His, Ile, Leu, Met, Phe, Trp, Val (GR1+ILV), and (4) Arg, His, Lys, Met, Phe, Thr, Trp (GR1+ALT). Within each AA infusion, EAA were infused in amounts relative to their content in 1 kg of casein, according to
      • Metcalf J.A.
      • Crompton L.A.
      • Wray-Cahen D.
      • Lomax M.A.
      • Sutton J.D.
      • Beever D.E.
      • MacRae J.C.
      • Bequette B.J.
      • Backwell F.R.C.
      • Lobley G.E.
      Responses in milk constituents to intravascular administration of two mixtures of amino acids to dairy cows.
      . Including intake from the restricted feeding level of the basal diet and the infusions, target requirements for NEL and MP were formulated to be met to 90 and 75%, respectively, for SAL, and 95 and 104%, respectively, for AA infusions. Treatment solutions were prepared in 15-L batches, which were stored at room temperature for no more than 48 h before administration. Treatments were replenished daily and infused via multichannel peristaltic pumps at a rate of 10.4 mL/min to facilitate 120 h of continuous infusion.
      Table 1Rate of AA infusion (g/d) into the abomasum of lactating dairy cattle over 5 d
      AA
      Anhydrous.
      Treatment
      Abomasal infusion treatments were saline (negative control) and 4 different AA profiles. SAL = 0.9% saline infusion; EAAC = a complete mixture of EAA (Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, and Val); ILV = mixture of Ile, Leu, and Val; GR1+ILV = mixture of His, Ile, Leu, Met, Phe, Trp, and Val; GR1+ALT = mixture of Arg, His, Lys, Met, Phe, Thr, and Trp. Amino acids within treatments were infused in amounts relative to their content in casein. All AA treatments supplied 562 g of MP/d, and solutions were delivered in 15-L batches.
      SALEAACILVGR1+ILVGR1+ALT
      l-Arg0390064
      l-His03204752
      l–Ile057150840
      l-Leu0942451380
      l-Lys09800159
      dl–Met02704044
      l-Phe0940138152
      l-Thr0420068
      l-Trp01402123
      l-Val064167940
      Total0562562562562
      1 Anhydrous.
      2 Abomasal infusion treatments were saline (negative control) and 4 different AA profiles. SAL = 0.9% saline infusion; EAAC = a complete mixture of EAA (Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, and Val); ILV = mixture of Ile, Leu, and Val; GR1+ILV = mixture of His, Ile, Leu, Met, Phe, Trp, and Val; GR1+ALT = mixture of Arg, His, Lys, Met, Phe, Thr, and Trp. Amino acids within treatments were infused in amounts relative to their content in casein. All AA treatments supplied 562 g of MP/d, and solutions were delivered in 15-L batches.

      Blood Sampling, Isotope Infusion, and Analysis

      At 0730, 0930, 1130, 1330, and 1530 h on d 4 of each period, blood samples were collected by venipuncture into 10 mL sodium heparin and potassium EDTA vacutainers (Becton Dickinson) from the coccygeal vessels and from the subcutaneous abdominal vein of each cow. Arteriovenous differences (AVD) across the tail are assumed to be negligible and thus samples from the coccygeal vessels are representative of mammary arterial supply (
      • Emery R.S.
      • Brown L.D.
      • Bell J.W.
      Correlation of milk fat with dietary and metabolic factors in cows fed restricted-roughage rations supplemented with magnesium oxide or sodium bicarbonate.
      ). Samples were collected from the left and right subcutaneous abdominal veins, alternating at each sampling time point, to account for differences between sides. Collection tubes were immediately placed in ice and centrifuged at 3,000 × g for 15 min at room temperature. Plasma from each time point was collected and stored at −80°C pending analysis of AA, peptides, and AA metabolites. Plasma for analysis of other metabolites was pooled over sampling time points by cow and period and stored at −20°C until analysis. Plasma AA, peptide, and AA metabolite concentrations were determined using an ultra-performance liquid chromatography-MS system (Waters Acquity Ultra Performance LC system, Waters Corp.) as described by
      • Haque M.N.
      • Rulquin H.
      • Andrade A.
      • Faverdin P.
      • Peyraud J.L.
      • Lemosquet S.
      Milk protein synthesis in response to the provision of an “ideal” amino acid profile at 2 levels of metabolizable protein supply in dairy cows.
      . Plasma concentrations of glucose, BHB, nonesterified fatty acids, triacylglycerol (TAG), and urea were analyzed as described by
      • Nichols K.
      • Bannink A.
      • Dijkstra J.
      Energy and nitrogen balance of dairy cattle as affected by provision of different essential amino acid profiles at the same metabolizable protein supply.
      .
      During the 5-d adaptation period in the CRC, cows were fitted with semi-permanent double-lumen catheters (length, 16 cm; o.d. 2.5 mm; distal lumen, i.d. 1.19 mm, o.d. 1.65 mm; proximal lumen, i.d. 0.84 mm, o.d. 1.27 mm; MultiCath 2, art. no. 157.167; Vygon) in the right jugular vein. Catheters were checked twice daily for patency and flushed with heparinized saline for the duration of the experiment. On d 5 of each period, D-[U-13C]glucose (3.5 mmol/h, 99 atom % 13C; Sigma Aldrich) was continuously infused into the right jugular vein of each cow for 520 min, preceded by a priming dose of 13 mmol. Blood was collected from the coccygeal vessels of each cow into 10 mL sodium heparin vacutainers (Becton Dickinson) by venipuncture at 0500 h to serve as a background sample. The infusion for individual cows began approximately 15 min after their morning milking was completed. Blood samples were collected from the coccygeal vessels of each cow 460 and 490 min after the infusion started. Collection tubes were immediately placed in ice and centrifuged at 3,000 × g for 15 min at room temperature. Plasma from each time point was collected and stored at −80°C. Additional milk samples were collected from each cow during milking on d 5 and stored at −20°C.
      Blood plasma (100 µL) was treated with 0.8 mL acetonitrile followed by centrifugation (3,000 × g for 10 min at room temperature), and the supernatant was evaporated at 45°C for 40 min under vacuum. Milk samples collected on d 5 were defatted, deproteinized, and hydrolyzed with β-galactosidase (EC 3.2.1.23) using the methods described by
      • Sunehag A.L.
      • Louie K.
      • Bier J.L.
      • Tigas S.
      • Haymond M.W.
      Hexoneogenesis in the human breast during lactation.
      . Hydrolyzed milk serum (50 µL) was dried under vacuum at 45°C for 2 h. According to the methods described by
      • Schierbeek H.
      • Moerdijk-Poortvliet T.C.W.
      • van den Akker C.H.P.
      • te Braake F.W.J.
      • Boschker H.T.S.
      • van Goudoever J.B.
      Analysis of [U-13C6] glucose in human plasma using liquid chromatography/isotope ratio mass spectrometry compared with two other mass spectrometry techniques.
      , aldonitrile pentaacetate derivatives of blood plasma and hydrolyzed milk serum were prepared by adding 100 µL of 2% hydroxylamine-HCl to pyridine and incubating together at 90°C for 30 min. Subsequently, 50 µL acetic anhydride was added and incubated at 90°C for 30 min. Remaining liquid was evaporated under a N stream at 50°C. Samples were solubilized with 100 µL chloroform and analyzed for 13C enrichment (in plasma glucose and in milk glucose and galactose) by GC combustion isotope ratio MS analysis using a DELTA V isotope ratio mass spectrometer (Thermo Fisher Scientific) coupled with a Trace Ultra GC (Thermo Fisher Scientific). A Zebron ZB-MultiResidue-1 capillary column (length, 30 m; i.d. 0.25 mm; film thickness 0.25 µm; Phenomenex) and splitless injection was used for the chromatographic separation. The temperature was held at 60°C for 0.05 min, then raised 14.5°C/min and held at 230°C for 6 min. After separation, the combustion phase used an oven temperature held at 60°C for 2 min, raised 25°C/min, and finally held at 230°C for 11.2 min. The flow of helium carrier gas was maintained at 2.0 mL/min.
      Gaseous exchange (CH4, O2, and CO2) in the CRC was measured as described by
      • Nichols K.
      • Bannink A.
      • Dijkstra J.
      Energy and nitrogen balance of dairy cattle as affected by provision of different essential amino acid profiles at the same metabolizable protein supply.
      . Concentrations of 13C were analyzed in outgoing air in 10-min intervals by nondispersive infrared spectrometry (Advance Optima Uras 14 NDIR analyzer; ABB) as described by
      • Alferink S.J.J.
      • van den Borne J.J.G.C.
      • Gerrits W.J.J.
      • Lammers-Wienhoven S.C.W.
      • Heetkamp M.J.W.
      On-line, continuous determination of 13CO2/12CO2 ratios by nondispersive infrared absorption in indirect calorimetry facilities.
      . The analyzer was faulty in one chamber and could not be repaired during the experiment, resulting in measurement of 13CO2 dynamics in 4 of 5 chambers per period.

      Calculations and Statistical Analysis

      Long-chain fatty acid concentrations were calculated on a molar basis as 3 × TAG + nonesterified fatty acids. Plasma concentrations of AA, peptides, and AA metabolites were averaged over the 5 sampling times on d 4. Milk CP was assumed to consist of 94.5% true protein (
      • DePeters E.J.
      • Ferguson J.D.
      Nonprotein nitrogen distribution in the milk of cows.
      ). All following calculations were based on this estimate of true protein yield. Mammary plasma flow (MPF) across the whole udder was estimated according to the Fick principle using Phe and Tyr as internal markers (
      • Cant J.P.
      • DePeters E.J.
      • Baldwin R.L.
      Mammary amino acid utilization in dairy cows fed fat and its relationship to milk protein depression.
      ), where MPF (L/h) = [milk Phe + Tyr output (μmol/h)]/[Phe + Tyr AVD (μmol/L)], with an allowance for 3.37% contribution of blood-derived proteins to milk Phe + Tyr (
      • Lapierre H.
      • Lobley G.E.
      • Doepel L.
      • Raggio G.
      • Rulquin H.
      • Lemosquet S.
      Triennial Lactation Symposium: Mammary metabolism of amino acids in dairy cows.
      ). Milk output of Phe + Tyr was estimated from the afternoon milk protein yield of d 4 of infusion, corresponding to the blood samples taken that day, using mean Phe and Tyr contents reported by
      • Mepham T.B.
      The composition of milks.
      and
      • Lapierre H.
      • Lobley G.E.
      • Doepel L.
      • Raggio G.
      • Rulquin H.
      • Lemosquet S.
      Triennial Lactation Symposium: Mammary metabolism of amino acids in dairy cows.
      . Uptakes (mmol/h) of metabolites across the mammary glands were calculated as the product of their plasma AVD and MPF. Positive uptake values indicate a net removal from plasma, whereas negative values indicate net release from the mammary glands. Mammary clearances were calculated from the model of
      • Hanigan M.D.
      • France J.
      • Wray-Cahen D.
      • Beever D.E.
      • Lobley G.E.
      • Reutzel L.
      • Smith N.E.
      Alternative models for analyses of liver and mammary transorgan metabolite extraction data.
      , where clearance (L/h) = (AVD × MPF)/venous concentration. The average milk protein AA composition of that reported by
      • Mepham T.B.
      The composition of milks.
      and
      • Lapierre H.
      • Lobley G.E.
      • Doepel L.
      • Raggio G.
      • Rulquin H.
      • Lemosquet S.
      Triennial Lactation Symposium: Mammary metabolism of amino acids in dairy cows.
      and milk protein yield from the afternoon milking on d 4 of infusion was used to calculate mammary gland AA uptake to milk true protein output ratios (U:O). Mammary gland glucose balance was calculated according to estimations of
      • Dijkstra J.
      • France J.
      • Assis A.G.
      • Neal H. D. St. C.
      • Campos O.F.
      • Aroeira L.M.J.
      Simulation of digestion in cattle fed sugarcane: Prediction of nutrient supply for milk production with locally available supplements.
      using milk lactose and fat yield from the afternoon milking on d 4 of infusion.
      Plasma samples collected after the start of tracer infusion were adjusted for background isotopic enrichment (IE) of the 0500 h sample to calculate atom percent excess (APE) of 13C in plasma glucose during the tracer infusion. Plasma APE was analyzed using the MIXED procedure of SAS (version 9.4; SAS Institute Inc.) for treatment, sampling time, and treatment × sampling time effects, with treatment and period as fixed effects, cow as a random effect, and sampling time as a repeated measure using a first-order autoregressive covariance structure. There was no treatment × sampling time effect on plasma APE (P = 0.84). Multiple comparisons between treatment least squares means for sampling time point determined that APE at 460 and 490 min after initiation of tracer infusion did not differ (P > 0.65). Therefore, the animals were considered to be in steady-state after 460 min of infusion, and the mean APE of the 2 samples were used to calculate WB Ra of glucose (mmol/h) using the steady-state model:
      WB Ra = F × [(IEinf/IEp) – 1],


      where F is the D-[U-13C]glucose infusion rate (3.5 mmol/h), IEinf is the IE of 13C in the infusate (99 atom %), and IEp is the IE of 13C in plasma glucose. Infusion of U-13C-glucose and measurement of total IE may result in an underestimation of WB Ra if isotopomers m+1, m+2, and m+3 formed during the infusion period are not measured; however, the formation of these glucose isotopomers in dairy cattle is minimal (H. Lapierre, Agriculture and Agri-Food Canada, Sherbrooke, Quebec, personal communication), and thus the quantification of total enrichment in this experiment is considered appropriate.
      Hourly IE of 13CO2 was calculated as the average of all 13C measurements within each hour. Hourly background IE of 13CO2 from d 4 (no tracer infusion) was used to calculate hourly 13CO2 production (the product of 13CO2 APE and hourly CO2 production) for a 32-h period (0000 h on d 5 through 0800 h on d 6) covering the period of tracer infusion (Figure 1). The positive incremental area under the curve for 13CO2 production (mmol) was evaluated using the trapezoid rule for values above baseline and was calculated for the 24 h period between 0500 h on d 5 and 0500 h on d 6. Production of 13CO2 over this 24 h period was used to calculate the recovery of infused 13C glucose in expired CO2. Assuming all 13C recovered in CO2 arose from Ra glucose, glucose oxidation rate (mmol/h) was calculated as the product of 13C recovery in CO2 (as a fraction of infused 13C) and WB Ra of glucose (mmol/h) after applying a correction factor of 0.70 to account for 13C sequestration in bicarbonate (
      • Junghans P.
      • Voigt J.
      • Jentsch W.
      • Metges C.C.
      • Derno M.
      The 13C bicarbonate dilution technique to determine energy expenditure in young bulls validated by indirect calorimetry.
      ).
      Figure thumbnail gr1
      Figure 1Production of 13CO2 (mmol/h) during a primed continuous infusion of D-[U-13C]glucose in lactating dairy cows receiving abomasal infusions of saline (SAL) or 562 g/d of AA in different profiles; SEM averaged 0.320, 0.278, 0.278, 0.320, and 0.278 for SAL (▪), EAAC (♦), ILV (▴), GR1+ILV (×), and GR1+ALT (•), respectively. n = 4 for all treatments except GR1+ALT, where n = 3. Arrows indicate the mean time of isotope infusion start and end across cow and period. The shaded area indicates the 24-h period (0500 h on d 5 to 0500 h on d 6) used for calculation of the incremental area under the curve and 13C recovery. SAL = 0.9% saline infusion; EAAC = a complete mixture of EAA (Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, and Val); ILV = mixture of Ile, Leu, and Val; GR1+ILV = mixture of His, Ile, Leu, Met, Phe, Trp, and Val; GR1+ALT = mixture of Arg, His, Lys, Met, Phe, Thr, and Trp. Amino acids within treatments were infused in amounts relative to their content in casein. All AA treatments supplied 562 g of MP/d, and solutions were delivered in 15-L batches.
      One cow did not receive the correct treatment in period 1 and was thus removed from the statistical analysis for this period (n = 4 for GR1+ALT; n = 5 for all other treatments). The jugular catheter of 1 cow lost patency in period 5 resulting in a lost observation for isotope-related measurements in this period. This cow was housed in the chamber with the faulty 13CO2 analyzer, resulting in no additional missing measurements of 13CO2 dynamics. Variances in milk yield and composition, plasma constituents, and parameters relating to mammary metabolism and WB glucose metabolism were analyzed using the MIXED procedure of SAS (version 9.4; SAS Institute Inc.). The model contained treatment and period as fixed effects and cow as a random effect. We observed no carryover effects between periods, assessed by testing for an effect of the previous treatment in the ANOVA. Differences were considered significant at P ≤ 0.05 and tendencies at 0.05 < P ≤ 0.10. Multiple comparisons between treatment means were made using the Tukey-Kramer method when a treatment effect was detected at P ≤ 0.05. Tendencies for differences between treatments are described in the Supplemental File S1 (https://data.mendeley.com/datasets/gxtcx8rn3w/1;
      • Nichols K.
      Supplementary data for “Essential amino acid profile of supplemental metabolizable protein affects mammary gland metabolism and whole-body glucose kinetics in dairy cattle” by Nichols et al. Mendeley Data, V12.
      ). Mammary clearance results for individual AA are described in Supplemental Table S1 (https://data.mendeley.com/datasets/gxtcx8rn3w/1;
      • Nichols K.
      Supplementary data for “Essential amino acid profile of supplemental metabolizable protein affects mammary gland metabolism and whole-body glucose kinetics in dairy cattle” by Nichols et al. Mendeley Data, V12.
      ). Arterial concentrations and mammary metabolism of glucose (beyond what is described below), urea, BHB, nonesterified fatty acids, TAG, and long-chain fatty acids is described in Supplemental Tables S2 and S3 (https://data.mendeley.com/datasets/gxtcx8rn3w/1;
      • Nichols K.
      Supplementary data for “Essential amino acid profile of supplemental metabolizable protein affects mammary gland metabolism and whole-body glucose kinetics in dairy cattle” by Nichols et al. Mendeley Data, V12.
      ).

      RESULTS

      Milk Production

      Daily lactation performance and DMI have been presented by
      • Nichols K.
      • Bannink A.
      • Dijkstra J.
      Energy and nitrogen balance of dairy cattle as affected by provision of different essential amino acid profiles at the same metabolizable protein supply.
      . Daily DMI averaged (mean ± SD) 19.9 ± 0.28 kg/d for SAL, EAAC, GR1+ILV, and GR1+ALT, and was 17.9 kg/d for ILV. The present paper reports milk production expressed on an hourly basis from the afternoon milking on d 4 and 5 of infusion (Table 2). Milk protein yield increased over SAL with EAAC and GR1+ILV (P ≤ 0.03). Infusion of ILV decreased milk protein yield compared with EAAC and GR1+ILV (P ≤ 0.04). Infusion of ILV increased milk fat content over EAAC (P = 0.03). Lactose content was lower on GR1+ILV infusion compared with SAL, ILV, and GR1+ALT (P ≤ 0.03), and lower on EAAC compared with ILV (P = 0.05).
      Table 2Milk and component production of lactating dairy cows receiving 5-d abomasal infusions of saline (SAL) or 562 g/d of AA as a complete mixture of EAA (EAAC), as a mixture of Ile, Leu, and Val (ILV), as a mixture of group 1 AA and Ile, Leu, and Val (GR1+ILV), and as a mixture of group 1 AA and Arg, Lys, and Thr (GR1+ALT)
      Data are least squares means from the afternoon milkings on d 4 and 5 of infusion.
      ItemTreatment
      Abomasal infusion treatments were saline (negative control) and 4 different AA profiles. SAL = 0.9% saline infusion; EAAC = a complete mixture of EAA (Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, and Val); ILV = mixture of Ile, Leu, and Val; GR1+ILV = mixture of His, Ile, Leu, Met, Phe, Trp, and Val; GR1+ALT = mixture of Arg, His, Lys, Met, Phe, Thr, and Trp. Amino acids within treatments were infused in amounts relative to their content in casein. All AA treatments supplied 562 g of MP/d, and solutions were delivered in 15-L batches.
      SEMP-value
      SALEAACILVGR1+ILVGR1+ALT
      Milk, kg/h1.271.491.351.491.420.0880.04
      CP, g/h39.1
      Means within a row with no common superscripts differ (P < 0.05).
      48.5
      Means within a row with no common superscripts differ (P < 0.05).
      39.3
      Means within a row with no common superscripts differ (P < 0.05).
      47.8
      Means within a row with no common superscripts differ (P < 0.05).
      44.6
      Means within a row with no common superscripts differ (P < 0.05).
      2.300.01
      CP, g/kg30.832.629.432.231.61.050.05
      Fat, g/h61.561.766.868.262.04.030.20
      Fat, g/kg48.4
      Means within a row with no common superscripts differ (P < 0.05).
      41.9
      Means within a row with no common superscripts differ (P < 0.05).
      50.8
      Means within a row with no common superscripts differ (P < 0.05).
      46.3
      Means within a row with no common superscripts differ (P < 0.05).
      44.9
      Means within a row with no common superscripts differ (P < 0.05).
      3.760.05
      Lactose, g/h60.068.663.767.966.94.020.11
      Lactose, g/kg47.1
      Means within a row with no common superscripts differ (P < 0.05).
      46.1
      Means within a row with no common superscripts differ (P < 0.05).
      47.3
      Means within a row with no common superscripts differ (P < 0.05).
      45.6
      Means within a row with no common superscripts differ (P < 0.05).
      47.0
      Means within a row with no common superscripts differ (P < 0.05).
      0.36<0.01
      a–c Means within a row with no common superscripts differ (P < 0.05).
      1 Data are least squares means from the afternoon milkings on d 4 and 5 of infusion.
      2 Abomasal infusion treatments were saline (negative control) and 4 different AA profiles. SAL = 0.9% saline infusion; EAAC = a complete mixture of EAA (Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, and Val); ILV = mixture of Ile, Leu, and Val; GR1+ILV = mixture of His, Ile, Leu, Met, Phe, Trp, and Val; GR1+ALT = mixture of Arg, His, Lys, Met, Phe, Thr, and Trp. Amino acids within treatments were infused in amounts relative to their content in casein. All AA treatments supplied 562 g of MP/d, and solutions were delivered in 15-L batches.

      Arterial Concentrations

      Arterial plasma concentration of EAA, group 1 AA, group 2 AA, and nonbranched (NB)-group 2 AA increased with EAAC infusion over SAL (P ≤ 0.05; Table 3). Individually, concentration of Arg, His, Lys, Met, Phe, and Trp increased over SAL with EAAC (P ≤ 0.02). Infusion of ILV increased arterial concentration of total AA (TAA) over SAL, EAAC, and GR1+ALT (P ≤ 0.03), and increased concentrations of EAA, group 2 AA, and branched-chain (BC)-group 2 AA (as a group and individually) over SAL and the other AA infusions (P ≤ 0.01). Group 1 AA concentrations (as a group and individually) were lower during ILV infusion compared with the other AA infusions (P ≤ 0.01), and did not differ from SAL (P ≥ 0.28). Concentration of NB-group 2 AA (as a group and individually) were lower during ILV infusion compared with EAAC and GR1+ALT (P ≤ 0.01). Infusion of GR1+ILV increased arterial concentration of TAA, EAA, group 1 AA, group 2 AA, and BC-group 2 AA over SAL (P ≤ 0.02), and increased group 1 AA concentration and decreased NB-group 2 AA concentrations (as a group and individually) compared with EAAC (P < 0.01). Infusion of GR1+ALT increased group 1 AA and NB-group 2 AA concentrations over SAL and EAAC (P < 0.01). In response to GR1+ILV and GR1+ALT, individual concentrations of all EAA included in the respective infusions increased over SAL (P ≤ 0.01), with the exception of Ile during GR1+ILV infusion. Infusion of GR1+ILV increased concentrations of His, Met, and Phe compared with EAAC (P ≤ 0.02). Infusion of GR1+ALT increased concentrations of Lys, Met, Phe, Thr, and Trp (P ≤ 0.04) and decreased the concentration of Val (P = 0.04) compared with EAAC. Concentration of BC-group 2 AA (as a group), Leu, and Val increased over GR1+ALT with GR1+ILV infusion (P < 0.01), and concentration of NB-group 2 AA (as a group and individually) increased over GR1+ILV with GR1+ALT infusion (P < 0.01).
      Table 3Arterial plasma concentrations (μM) of AA, peptides, and AA metabolites in lactating dairy cows receiving 5-d abomasal infusions of saline (SAL) or 562 g/d of AA as a complete mixture of EAA (EAAC), as a mixture of Ile, Leu, and Val (ILV), as a mixture of group 1 AA and Ile, Leu, and Val (GR1+ILV), and as a mixture of group 1 AA and Arg, Lys, and Thr (GR1+ALT)
      ItemTreatment
      Abomasal infusion treatments were saline (negative control) and 4 different AA profiles. SAL = 0.9% saline infusion; EAAC = a complete mixture of EAA (Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, and Val); ILV = mixture of Ile, Leu, and Val; GR1+ILV = mixture of His, Ile, Leu, Met, Phe, Trp, and Val; GR1+ALT = mixture of Arg, His, Lys, Met, Phe, Thr, and Trp. Amino acids within treatments were infused in amounts relative to their content in casein. All AA treatments supplied 562 g of MP/d, and solutions were delivered in 15-L batches.
      SEMP-value
      SALEAACILVGR1+ILVGR1+ALT
      EAA
      EAA = Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Val.
      670
      Means within a row with no common superscripts differ (P < 0.05).
      1,351
      Means within a row with no common superscripts differ (P < 0.05).
      2,174
      Means within a row with no common superscripts differ (P < 0.05).
      1,545
      Means within a row with no common superscripts differ (P < 0.05).
      1,117
      Means within a row with no common superscripts differ (P < 0.05).
      147.8<0.01
      Group 1
      Group 1 = His, Met, Phe+Tyr, Trp.
      147
      Means within a row with no common superscripts differ (P < 0.05).
      274
      Means within a row with no common superscripts differ (P < 0.05).
      116
      Means within a row with no common superscripts differ (P < 0.05).
      430
      Means within a row with no common superscripts differ (P < 0.05).
      409
      Means within a row with no common superscripts differ (P < 0.05).
      12.2<0.01
      Group 2
      Group 2 = Arg, Ile, Leu, Lys, Thr, Val.
      556
      Means within a row with no common superscripts differ (P < 0.05).
      1,111
      Means within a row with no common superscripts differ (P < 0.05).
      2,080
      Means within a row with no common superscripts differ (P < 0.05).
      1,182
      Means within a row with no common superscripts differ (P < 0.05).
      772
      Means within a row with no common superscripts differ (P < 0.05).
      146.1<0.01
      BC-Group 2
      BC-Group 2 = Ile, Leu, Val.
      354
      Means within a row with no common superscripts differ (P < 0.05).
      753
      Means within a row with no common superscripts differ (P < 0.05).
      1,933
      Means within a row with no common superscripts differ (P < 0.05).
      1,021
      Means within a row with no common superscripts differ (P < 0.05).
      234
      Means within a row with no common superscripts differ (P < 0.05).
      141.2<0.01
      NB-Group 2
      NB-Group 2 = Arg, Lys, Thr.
      202
      Means within a row with no common superscripts differ (P < 0.05).
      358
      Means within a row with no common superscripts differ (P < 0.05).
      148
      Means within a row with no common superscripts differ (P < 0.05).
      161
      Means within a row with no common superscripts differ (P < 0.05).
      539
      Means within a row with no common superscripts differ (P < 0.05).
      24.6<0.01
      NEAA
      NEAA = Ala, Asn, Asp, Cit, Cys, Gln, Glu, Gly, Orn, Pro, Ser, Tyr.
      1,1609481,0541,1331,19958.10.07
      TAA
      TAA = EAA + NEAA.
      1,831
      Means within a row with no common superscripts differ (P < 0.05).
      2,299
      Means within a row with no common superscripts differ (P < 0.05).
      3,228
      Means within a row with no common superscripts differ (P < 0.05).
      2,678
      Means within a row with no common superscripts differ (P < 0.05).
      2,352
      Means within a row with no common superscripts differ (P < 0.05).
      169.4<0.01
      Arg55
      Means within a row with no common superscripts differ (P < 0.05).
      90
      Means within a row with no common superscripts differ (P < 0.05).
      50
      Means within a row with no common superscripts differ (P < 0.05).
      55
      Means within a row with no common superscripts differ (P < 0.05).
      118
      Means within a row with no common superscripts differ (P < 0.05).
      6.8<0.01
      His23
      Means within a row with no common superscripts differ (P < 0.05).
      60
      Means within a row with no common superscripts differ (P < 0.05).
      20
      Means within a row with no common superscripts differ (P < 0.05).
      83
      Means within a row with no common superscripts differ (P < 0.05).
      75
      Means within a row with no common superscripts differ (P < 0.05).
      5.0<0.01
      Ile106
      Means within a row with no common superscripts differ (P < 0.05).
      169
      Means within a row with no common superscripts differ (P < 0.05).
      363
      Means within a row with no common superscripts differ (P < 0.05).
      207
      Means within a row with no common superscripts differ (P < 0.05).
      93
      Means within a row with no common superscripts differ (P < 0.05).
      30.4<0.01
      Leu89
      Means within a row with no common superscripts differ (P < 0.05).
      206
      Means within a row with no common superscripts differ (P < 0.05).
      588
      Means within a row with no common superscripts differ (P < 0.05).
      297
      Means within a row with no common superscripts differ (P < 0.05).
      60
      Means within a row with no common superscripts differ (P < 0.05).
      44.2<0.01
      Lys54
      Means within a row with no common superscripts differ (P < 0.05).
      116
      Means within a row with no common superscripts differ (P < 0.05).
      37
      Means within a row with no common superscripts differ (P < 0.05).
      48
      Means within a row with no common superscripts differ (P < 0.05).
      175
      Means within a row with no common superscripts differ (P < 0.05).
      9.1<0.01
      Met17
      Means within a row with no common superscripts differ (P < 0.05).
      48
      Means within a row with no common superscripts differ (P < 0.05).
      11
      Means within a row with no common superscripts differ (P < 0.05).
      91
      Means within a row with no common superscripts differ (P < 0.05).
      77
      Means within a row with no common superscripts differ (P < 0.05).
      3.1<0.01
      Phe46
      Means within a row with no common superscripts differ (P < 0.05).
      94
      Means within a row with no common superscripts differ (P < 0.05).
      36
      Means within a row with no common superscripts differ (P < 0.05).
      145
      Means within a row with no common superscripts differ (P < 0.05).
      145
      Means within a row with no common superscripts differ (P < 0.05).
      8.6<0.01
      Thr93
      Means within a row with no common superscripts differ (P < 0.05).
      151
      Means within a row with no common superscripts differ (P < 0.05).
      61
      Means within a row with no common superscripts differ (P < 0.05).
      58
      Means within a row with no common superscripts differ (P < 0.05).
      245
      Means within a row with no common superscripts differ (P < 0.05).
      13.8<0.01
      Trp29
      Means within a row with no common superscripts differ (P < 0.05).
      38
      Means within a row with no common superscripts differ (P < 0.05).
      27
      Means within a row with no common superscripts differ (P < 0.05).
      45
      Means within a row with no common superscripts differ (P < 0.05).
      47
      Means within a row with no common superscripts differ (P < 0.05).
      2.4<0.01
      Val160
      Means within a row with no common superscripts differ (P < 0.05).
      378
      Means within a row with no common superscripts differ (P < 0.05).
      982
      Means within a row with no common superscripts differ (P < 0.05).
      517
      Means within a row with no common superscripts differ (P < 0.05).
      105
      Means within a row with no common superscripts differ (P < 0.05).
      68.2<0.01
      Ala220
      Means within a row with no common superscripts differ (P < 0.05).
      162
      Means within a row with no common superscripts differ (P < 0.05).
      128
      Means within a row with no common superscripts differ (P < 0.05).
      196
      Means within a row with no common superscripts differ (P < 0.05).
      205
      Means within a row with no common superscripts differ (P < 0.05).
      16.3<0.01
      Asn42364043422.90.51
      Asp15
      Means within a row with no common superscripts differ (P < 0.05).
      13
      Means within a row with no common superscripts differ (P < 0.05).
      11
      Means within a row with no common superscripts differ (P < 0.05).
      16
      Means within a row with no common superscripts differ (P < 0.05).
      15
      Means within a row with no common superscripts differ (P < 0.05).
      1.00.02
      Cit57
      Means within a row with no common superscripts differ (P < 0.05).
      75
      Means within a row with no common superscripts differ (P < 0.05).
      88
      Means within a row with no common superscripts differ (P < 0.05).
      84
      Means within a row with no common superscripts differ (P < 0.05).
      68
      Means within a row with no common superscripts differ (P < 0.05).
      9.30.05
      Cys3.9
      Means within a row with no common superscripts differ (P < 0.05).
      6.1
      Means within a row with no common superscripts differ (P < 0.05).
      2.7
      Means within a row with no common superscripts differ (P < 0.05).
      5.9
      Means within a row with no common superscripts differ (P < 0.05).
      6.6
      Means within a row with no common superscripts differ (P < 0.05).
      0.630.01
      Gln251
      Means within a row with no common superscripts differ (P < 0.05).
      183
      Means within a row with no common superscripts differ (P < 0.05).
      289
      Means within a row with no common superscripts differ (P < 0.05).
      222
      Means within a row with no common superscripts differ (P < 0.05).
      208
      Means within a row with no common superscripts differ (P < 0.05).
      18.00.01
      Glu38333332373.10.32
      Gly31124127027831322.80.11
      Orn25
      Means within a row with no common superscripts differ (P < 0.05).
      41
      Means within a row with no common superscripts differ (P < 0.05).
      22
      Means within a row with no common superscripts differ (P < 0.05).
      26
      Means within a row with no common superscripts differ (P < 0.05).
      62
      Means within a row with no common superscripts differ (P < 0.05).
      2.5<0.01
      Pro69605574754.90.05
      Ser94
      Means within a row with no common superscripts differ (P < 0.05).
      65
      Means within a row with no common superscripts differ (P < 0.05).
      94
      Means within a row with no common superscripts differ (P < 0.05).
      90
      Means within a row with no common superscripts differ (P < 0.05).
      101
      Means within a row with no common superscripts differ (P < 0.05).
      6.30.01
      Tyr33
      Means within a row with no common superscripts differ (P < 0.05).
      34
      Means within a row with no common superscripts differ (P < 0.05).
      22
      Means within a row with no common superscripts differ (P < 0.05).
      67
      Means within a row with no common superscripts differ (P < 0.05).
      66
      Means within a row with no common superscripts differ (P < 0.05).
      2.6<0.01
      Other AA, peptides, and AA metabolites
      Other N derivatives measured with ultra-performance liquid chromatography-mass spectrometry showing a plasma concentration higher than the limit of quantification.
       1 Methyl-histidine3.8
      Means within a row with no common superscripts differ (P < 0.05).
      3.0
      Means within a row with no common superscripts differ (P < 0.05).
      2.4
      Means within a row with no common superscripts differ (P < 0.05).
      2.9
      Means within a row with no common superscripts differ (P < 0.05).
      2.7
      Means within a row with no common superscripts differ (P < 0.05).
      0.34<0.01
       3 Methyl-histidine3.93.03.03.82.70.350.19
       α-Amino-n-butyric acid11
      Means within a row with no common superscripts differ (P < 0.05).
      22
      Means within a row with no common superscripts differ (P < 0.05).
      13
      Means within a row with no common superscripts differ (P < 0.05).
      22
      Means within a row with no common superscripts differ (P < 0.05).
      32
      Means within a row with no common superscripts differ (P < 0.05).
      2.5<0.01
       α-Amino-adipic acid2.9
      Means within a row with no common superscripts differ (P < 0.05).
      6.0
      Means within a row with no common superscripts differ (P < 0.05).
      1.8
      Means within a row with no common superscripts differ (P < 0.05).
      1.2
      Means within a row with no common superscripts differ (P < 0.05).
      9.1
      Means within a row with no common superscripts differ (P < 0.05).
      0.54<0.01
       β-Alanine3.73.83.83.92.90.290.20
       Carnosine911912121.10.05
       Cystathionine1.7
      Means within a row with no common superscripts differ (P < 0.05).
      2.3
      Means within a row with no common superscripts differ (P < 0.05).
      0.6
      Means within a row with no common superscripts differ (P < 0.05).
      3.8
      Means within a row with no common superscripts differ (P < 0.05).
      5.3
      Means within a row with no common superscripts differ (P < 0.05).
      0.31<0.01
       Hydroxylysine0.480.440.270.440.400.0850.37
       Hydroxyproline11
      Means within a row with no common superscripts differ (P < 0.05).
      10
      Means within a row with no common superscripts differ (P < 0.05).
      8
      Means within a row with no common superscripts differ (P < 0.05).
      12
      Means within a row with no common superscripts differ (P < 0.05).
      10
      Means within a row with no common superscripts differ (P < 0.05).
      0.90.03
       Phosphoserine0.520.570.820.770.580.0810.07
       Taurine29
      Means within a row with no common superscripts differ (P < 0.05).
      29
      Means within a row with no common superscripts differ (P < 0.05).
      26
      Means within a row with no common superscripts differ (P < 0.05).
      45
      Means within a row with no common superscripts differ (P < 0.05).
      50
      Means within a row with no common superscripts differ (P < 0.05).
      3.2<0.01
      a-d Means within a row with no common superscripts differ (P < 0.05).
      1 Abomasal infusion treatments were saline (negative control) and 4 different AA profiles. SAL = 0.9% saline infusion; EAAC = a complete mixture of EAA (Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, and Val); ILV = mixture of Ile, Leu, and Val; GR1+ILV = mixture of His, Ile, Leu, Met, Phe, Trp, and Val; GR1+ALT = mixture of Arg, His, Lys, Met, Phe, Thr, and Trp. Amino acids within treatments were infused in amounts relative to their content in casein. All AA treatments supplied 562 g of MP/d, and solutions were delivered in 15-L batches.
      2 EAA = Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Val.
      3 Group 1 = His, Met, Phe+Tyr, Trp.
      4 Group 2 = Arg, Ile, Leu, Lys, Thr, Val.
      5 BC-Group 2 = Ile, Leu, Val.
      6 NB-Group 2 = Arg, Lys, Thr.
      7 NEAA = Ala, Asn, Asp, Cit, Cys, Gln, Glu, Gly, Orn, Pro, Ser, Tyr.
      8 TAA = EAA + NEAA.
      9 Other N derivatives measured with ultra-performance liquid chromatography-mass spectrometry showing a plasma concentration higher than the limit of quantification.
      Infusion of EAAC increased arterial concentration of Orn (P < 0.01) and decreased concentration of Ser (P = 0.02) compared with SAL. Infusion of ILV increased concentration of Cit (P = 0.04) and decreased concentrations of Ala and Tyr (P ≤ 0.04) compared with SAL. In response to ILV, concentration of Cys and Tyr decreased compared with all other AA infusions (P ≤ 0.03), Ser concentration increased over EAAC (P = 0.01), Ala and Asp concentration decreased compared with GR1+ILV (P ≤ 0.03), and Ala concentration decreased compared with GR1+ALT (P = 0.02). Concentration of Gln increased with ILV over EAAC (P = 0.01) and concentration of Orn decreased with ILV compared with EAAC and GR1+ALT (P < 0.01). Compared with SAL, infusion of GR1+ILV increased concentration of Tyr (P < 0.01) and GR1+ALT infusion increased concentration of Orn and Tyr (P < 0.01). Compared with EAAC, concentrations of Ser and Tyr increased with GR1+ILV and GR1+ALT (P ≤ 0.04), and concentration of Orn decreased with GR1+ILV and increased with GR1+ALT (P ≤ 0.01). Concentration of Orn increased over GR1+ILV with infusion of GR1+ALT (P < 0.01).
      Arterial plasma concentration of 1 methyl-histidine decreased with ILV, GR1+ILV, and GR1+ALT (P ≤ 0.03) compared with SAL. Concentration of α-amino-n-butyric acid increased over SAL with EAAC, GR1+ILV and GR1+ALT infusions (P ≤ 0.05), and increased over ILV (P < 0.01) with GR1+ALT infusion. Concentration of α-amino-adipic acid increased over SAL, ILV, and GR1+ILV with infusion of EAAC and GR1+ALT (P ≤ 0.01), and increased over EAAC with GR1+ALT infusion (P = 0.01). Cystathionine concentration decreased with ILV infusion compared with all other AA infusions (P ≤ 0.01), increased with GR1+ILV and GR1+ALT infusions over SAL and EAAC (P ≤ 0.02), and increased with GR1+ALT infusion compared with GR1+ILV (P = 0.03). Infusion of ILV decreased hydroxyproline concentration compared with GR1+ILV (P = 0.02). Infusion of GR1+ILV and GR1+ALT increased concentration of taurine over SAL, EAAC, and ILV (P ≤ 0.02).

      Mammary Arteriovenous Differences

      Compared with SAL, infusion of EAAC increased AVD of EAA, group 2 AA, BC-group 2 AA, and NB-group 2 AA (P ≤ 0.02; Table 4). Individual AVD of His, Ile, Leu, and Lys increased (P ≤ 0.04) with EAAC over SAL. Infusion of ILV increased AVD of group 2 AA (P = 0.05) and BC-group 2 AA (as a group and individually; P < 0.05), and decreased AVD of Lys (P = 0.01) compared with SAL. Arteriovenous difference of NB-group 2 AA decreased with ILV compared with SAL and all AA infusions (P ≤ 0.03). Compared with all AA infusions, ILV decreased AVD of group 1 AA, and individual AVD of Arg, His, Lys, Met, and Thr (P ≤ 0.04). Infusion of ILV increased AVD of BC-group 2 AA (as a group and individually) compared with GR1+ALT (P ≤ 0.02), and increased AVD of Val compared with EAAC (P = 0.02). Infusion of GR1+ILV increased AVD of EAA, group 2 AA, and BC-group 2 AA (P < 0.01) compared with SAL. Individual AVD of His, Ile, Leu, Phe, and Val increased with GR1+ILV (P ≤ 0.03) compared with SAL. Compared with EAAC, infusion of GR1+ILV increased AVD of BC-group 2 AA (as a group and individually; P ≤ 0.04) and decreased AVD of NB-group 2 AA and Lys (P ≤ 0.05). Infusion of GR1+ALT increased AVD of NB-group 2 AA and Lys compared with SAL (P < 0.01). Arteriovenous difference of BC-group 2 AA (as a group and individually) increased over GR1+ALT with GR1+ILV infusion (P ≤ 0.01), and AVD of NB-group 2 AA and Lys increased over GR1+ILV with GR1+ALT infusion (P < 0.01).
      Table 4Mammary gland arteriovenous differences (μM) of AA in lactating dairy cows receiving 5-d abomasal infusions of saline (SAL) or 562 g/d of AA as a complete mixture of EAA (EAAC), as a mixture of Ile, Leu, and Val (ILV), as a mixture of group 1 AA and Ile, Leu, and Val (GR1+ILV), and as a mixture of group 1 AA and Arg, Lys, and Thr (GR1+ALT)
      AATreatment
      Abomasal infusion treatments were saline (negative control) and 4 different AA profiles. SAL = 0.9% saline infusion; EAAC = a complete mixture of EAA (Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, and Val); ILV = mixture of Ile, Leu, and Val; GR1+ILV = mixture of His, Ile, Leu, Met, Phe, Trp, and Val; GR1+ALT = mixture of Arg, His, Lys, Met, Phe, Thr, and Trp. Amino acids within treatments were infused in amounts relative to their content in casein. All AA treatments supplied 562 g of MP/d, and solutions were delivered in 15-L batches.
      SEMP-value
      SALEAACILVGR1+ILVGR1+ALT
      EAA
      EAA = Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Val.
      223
      Means within a row with no common superscripts differ (P < 0.05).
      299
      Means within a row with no common superscripts differ (P < 0.05).
      261
      Means within a row with no common superscripts differ (P < 0.05).
      323
      Means within a row with no common superscripts differ (P < 0.05).
      277
      Means within a row with no common superscripts differ (P < 0.05).
      16.6<0.01
      Group 1
      Group 1 = His, Met, Phe+Tyr, Trp.
      52
      Means within a row with no common superscripts differ (P < 0.05).
      67
      Means within a row with no common superscripts differ (P < 0.05).
      41
      Means within a row with no common superscripts differ (P < 0.05).
      68
      Means within a row with no common superscripts differ (P < 0.05).
      59
      Means within a row with no common superscripts differ (P < 0.05).
      3.8<0.01
      Group 2
      Group 2 = Arg, Ile, Leu, Lys, Thr, Val.
      185
      Means within a row with no common superscripts differ (P < 0.05).
      250
      Means within a row with no common superscripts differ (P < 0.05).
      231
      Means within a row with no common superscripts differ (P < 0.05).
      270
      Means within a row with no common superscripts differ (P < 0.05).
      233
      Means within a row with no common superscripts differ (P < 0.05).
      15.1<0.01
      BC-Group 2
      BC-Group 2 = Ile, Leu, Val.
      104
      Means within a row with no common superscripts differ (P < 0.05).
      144
      Means within a row with no common superscripts differ (P < 0.05).
      170
      Means within a row with no common superscripts differ (P < 0.05).
      183
      Means within a row with no common superscripts differ (P < 0.05).
      119
      Means within a row with no common superscripts differ (P < 0.05).
      12.1<0.01
      NB-Group 2
      NB-Group 2 = Arg, Lys, Thr.
      81
      Means within a row with no common superscripts differ (P < 0.05).
      106
      Means within a row with no common superscripts differ (P < 0.05).
      61
      Means within a row with no common superscripts differ (P < 0.05).
      87
      Means within a row with no common superscripts differ (P < 0.05).
      113
      Means within a row with no common superscripts differ (P < 0.05).
      4.4<0.01
      NEAA
      NEAA = Ala, Asn, Asp, Cit, Cys, Gln, Glu, Gly, Orn, Pro, Ser, Tyr.
      19717611917617318.80.10
      TAA
      TAA = EAA + NEAA.
      42047538049844330.30.08
      Arg25
      Means within a row with no common superscripts differ (P < 0.05).
      31
      Means within a row with no common superscripts differ (P < 0.05).
      21
      Means within a row with no common superscripts differ (P < 0.05).
      29
      Means within a row with no common superscripts differ (P < 0.05).
      31
      Means within a row with no common superscripts differ (P < 0.05).
      1.1<0.01
      His10
      Means within a row with no common superscripts differ (P < 0.05).
      12
      Means within a row with no common superscripts differ (P < 0.05).
      8
      Means within a row with no common superscripts differ (P < 0.05).
      12
      Means within a row with no common superscripts differ (P < 0.05).
      11
      Means within a row with no common superscripts differ (P < 0.05).
      0.5<0.01
      Ile27
      Means within a row with no common superscripts differ (P < 0.05).
      39
      Means within a row with no common superscripts differ (P < 0.05).
      42
      Means within a row with no common superscripts differ (P < 0.05).
      49
      Means within a row with no common superscripts differ (P < 0.05).
      32
      Means within a row with no common superscripts differ (P < 0.05).
      3.3<0.01
      Leu40
      Means within a row with no common superscripts differ (P < 0.05).
      59
      Means within a row with no common superscripts differ (P < 0.05).
      64
      Means within a row with no common superscripts differ (P < 0.05).
      72
      Means within a row with no common superscripts differ (P < 0.05).
      46
      Means within a row with no common superscripts differ (P < 0.05).
      4.2<0.01
      Lys35
      Means within a row with no common superscripts differ (P < 0.05).
      48
      Means within a row with no common superscripts differ (P < 0.05).
      23
      Means within a row with no common superscripts differ (P < 0.05).
      34
      Means within a row with no common superscripts differ (P < 0.05).
      56
      Means within a row with no common superscripts differ (P < 0.05).
      2.6<0.01
      Met9
      Means within a row with no common superscripts differ (P < 0.05).
      12
      Means within a row with no common superscripts differ (P < 0.05).
      7
      Means within a row with no common superscripts differ (P < 0.05).
      12
      Means within a row with no common superscripts differ (P < 0.05).
      10
      Means within a row with no common superscripts differ (P < 0.05).
      0.7<0.01
      Phe16
      Means within a row with no common superscripts differ (P < 0.05).
      22
      Means within a row with no common superscripts differ (P < 0.05).
      13
      Means within a row with no common superscripts differ (P < 0.05).
      23
      Means within a row with no common superscripts differ (P < 0.05).
      19
      Means within a row with no common superscripts differ (P < 0.05).
      1.5<0.01
      Thr22
      Means within a row with no common superscripts differ (P < 0.05).
      27
      Means within a row with no common superscripts differ (P < 0.05).
      16
      Means within a row with no common superscripts differ (P < 0.05).
      25
      Means within a row with no common superscripts differ (P < 0.05).
      27
      Means within a row with no common superscripts differ (P < 0.05).
      1.4<0.01
      Trp3.54.23.04.73.70.390.07
      Val36
      Means within a row with no common superscripts differ (P < 0.05).
      46
      Means within a row with no common superscripts differ (P < 0.05).
      64
      Means within a row with no common superscripts differ (P < 0.05).
      62
      Means within a row with no common superscripts differ (P < 0.05).
      41
      Means within a row with no common superscripts differ (P < 0.05).
      4.9<0.01
      Ala34
      Means within a row with no common superscripts differ (P < 0.05).
      8
      Means within a row with no common superscripts differ (P < 0.05).
      14
      Means within a row with no common superscripts differ (P < 0.05).
      6
      Means within a row with no common superscripts differ (P < 0.05).
      10
      Means within a row with no common superscripts differ (P < 0.05).
      5.80.03
      Asn9
      Means within a row with no common superscripts differ (P < 0.05).
      13
      Means within a row with no common superscripts differ (P < 0.05).
      5
      Means within a row with no common superscripts differ (P < 0.05).
      11
      Means within a row with no common superscripts differ (P < 0.05).
      11
      Means within a row with no common superscripts differ (P < 0.05).
      1.0<0.01
      Asp1.60.10.81.21.80.380.06
      Cit1.80.32.72.51.50.690.14
      Cys0.16
      Means within a row with no common superscripts differ (P < 0.05).
      0.49
      Means within a row with no common superscripts differ (P < 0.05).
      0.19
      Means within a row with no common superscripts differ (P < 0.05).
      0.79
      Means within a row with no common superscripts differ (P < 0.05).
      0.33
      Means within a row with no common superscripts differ (P < 0.05).
      0.1280.03
      Gln54
      Means within a row with no common superscripts differ (P < 0.05).
      59
      Means within a row with no common superscripts differ (P < 0.05).
      31
      Means within a row with no common superscripts differ (P < 0.05).
      65
      Means within a row with no common superscripts differ (P < 0.05).
      58
      Means within a row with no common superscripts differ (P < 0.05).
      6.40.02
      Glu28232122282.50.11
      Gly19131213124.40.72
      Orn13
      Means within a row with no common superscripts differ (P < 0.05).
      14
      Means within a row with no common superscripts differ (P < 0.05).
      11
      Means within a row with no common superscripts differ (P < 0.05).
      13
      Means within a row with no common superscripts differ (P < 0.05).
      19
      Means within a row with no common superscripts differ (P < 0.05).
      1.4<0.01
      Pro11
      Means within a row with no common superscripts differ (P < 0.05).
      8
      Means within a row with no common superscripts differ (P < 0.05).
      6
      Means within a row with no common superscripts differ (P < 0.05).
      8
      Means within a row with no common superscripts differ (P < 0.05).
      8
      Means within a row with no common superscripts differ (P < 0.05).
      0.90.05
      Ser1120516104.40.06
      Tyr14
      Means within a row with no common superscripts differ (P < 0.05).
      17
      Means within a row with no common superscripts differ (P < 0.05).
      11
      Means within a row with no common superscripts differ (P < 0.05).
      16
      Means within a row with no common superscripts differ (P < 0.05).
      15
      Means within a row with no common superscripts differ (P < 0.05).
      1.10.01
      a–d Means within a row with no common superscripts differ (P < 0.05).
      1 Abomasal infusion treatments were saline (negative control) and 4 different AA profiles. SAL = 0.9% saline infusion; EAAC = a complete mixture of EAA (Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, and Val); ILV = mixture of Ile, Leu, and Val; GR1+ILV = mixture of His, Ile, Leu, Met, Phe, Trp, and Val; GR1+ALT = mixture of Arg, His, Lys, Met, Phe, Thr, and Trp. Amino acids within treatments were infused in amounts relative to their content in casein. All AA treatments supplied 562 g of MP/d, and solutions were delivered in 15-L batches.
      2 EAA = Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Val.
      3 Group 1 = His, Met, Phe+Tyr, Trp.
      4 Group 2 = Arg, Ile, Leu, Lys, Thr, Val.
      5 BC-Group 2 = Ile, Leu, Val.
      6 NB-Group 2 = Arg, Lys, Thr.
      7 NEAA = Ala, Asn, Asp, Cit, Cys, Gln, Glu, Gly, Orn, Pro, Ser, Tyr.
      8 TAA = EAA + NEAA.
      Infusion of ILV decreased AVD of Pro (P = 0.03) compared with SAL, decreased AVD of Asn and Gln compared with EAAC and GR1+ILV (P ≤ 0.05), and decreased AVD of Asn compared with GR1+ALT (P = 0.02). Further, infusion of ILV decreased AVD of Tyr compared with EAAC and GR1+ILV (P ≤ 0.02), and decreased AVD of Cys compared with GR1+ILV (P = 0.04). Infusion of GR1+ILV decreased AVD of Ala (P = 0.03) and increased AVD of Cys (P = 0.03) compared with SAL. Infusion of GR1+ALT increased AVD of Orn over SAL, ILV, and GR1+ILV (P ≤ 0.02).

      Mammary Plasma Flow and AA Uptake

      Mammary plasma flow was faster on ILV infusion compared with EAAC and GR1+ILV (P ≤ 0.05; Table 5). Infusion of EAAC increased uptake of Leu and Phe compared with SAL (P ≤ 0.04). Infusion of ILV increased mammary uptake of EAA, group 2 AA, and BC-group 2 AA (as a group and individually) over SAL (P < 0.01). Infusion of ILV increased uptake of BC-group 2 AA, Leu, and Val over all AA infusions (P ≤ 0.03), increased uptake of Ile and decreased uptake of Lys compared with EAAC and GR1+ALT (P ≤ 0.01), decreased Phe uptake compared with EAAC and GR1+ILV (P ≤ 0.01), and decreased Thr uptake compared with GR1+ALT (P = 0.03). Infusion of GR1+ILV increased mammary uptake of EAA and BC-group 2 AA (P ≤ 0.04) over SAL. Individual uptakes of Ile, Leu, and Phe increased (P < 0.01) with GR1+ILV infusion over SAL. Uptake of Lys decreased (P = 0.02) with GR1+ILV compared with EAAC. Infusion of GR1+ALT increased uptake of NB-group 2 AA, Lys, and Thr over SAL (P ≤ 0.04) and increased uptake of NB-group 2 AA over ILV and GR1+ILV (P < 0.01). Uptake of Ile and Leu increased (P ≤ 0.02) with GR1+ILV over GR1+ALT. Uptake of Lys was higher (P < 0.01) on GR1+ALT compared with GR1+ILV. Infusion of EAAC decreased mammary uptake of Ala and Pro (P ≤ 0.04) compared with SAL. Infusion of ILV decreased uptake of Asn compared with EAAC and GR1+ALT (P ≤ 0.05), and increased uptake of Cit compared with EAAC (P = 0.04). Infusion of GR1+ILV decreased mammary uptake of Ala and Pro and increased uptake of Cys compared with SAL (P ≤ 0.03).
      Table 5Whole-mammary gland plasma flow and net AA uptakes in lactating dairy cows receiving 5-d abomasal infusions of saline (SAL) or 562 g/d of AA as a complete mixture of EAA (EAAC), as a mixture of Ile, Leu, and Val (ILV), as a mixture of group 1 AA and Ile, Leu, and Val (GR1+ILV), and as a mixture of group 1 AA and Arg, Lys, and Thr (GR1+ALT)
      ItemTreatment
      Abomasal infusion treatments were saline (negative control) and 4 different AA profiles. SAL = 0.9% saline infusion; EAAC = a complete mixture of EAA (Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, and Val); ILV = mixture of Ile, Leu, and Val; GR1+ILV = mixture of His, Ile, Leu, Met, Phe, Trp, and Val; GR1+ALT = mixture of Arg, His, Lys, Met, Phe, Thr, and Trp. Amino acids within treatments were infused in amounts relative to their content in casein. All AA treatments supplied 562 g of MP/d, and solutions were delivered in 15-L batches.
      SEMP-value
      SALEAACILVGR1+ILVGR1+ALT
      Plasma flow, L/h768
      Means within a row with no common superscripts differ (P < 0.05).
      703
      Means within a row with no common superscripts differ (P < 0.05).
      965
      Means within a row with no common superscripts differ (P < 0.05).
      693
      Means within a row with no common superscripts differ (P < 0.05).
      767
      Means within a row with no common superscripts differ (P < 0.05).
      70.10.04
      Net mammary uptake, mmol/h
       EAA
      EAA = Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Val.
      167
      Means within a row with no common superscripts differ (P < 0.05).
      209
      Means within a row with no common superscripts differ (P < 0.05).
      248
      Means within a row with no common superscripts differ (P < 0.05).
      219
      Means within a row with no common superscripts differ (P < 0.05).
      210
      Means within a row with no common superscripts differ (P < 0.05).
      11.8<0.01
       Group 1
      Group 1 = His, Met, Phe+Tyr, Trp.
      38.746.439.346.244.72.300.02
       Group 2
      Group 2 = Arg, Ile, Leu, Lys, Thr, Val.
      139
      Means within a row with no common superscripts differ (P < 0.05).
      174
      Means within a row with no common superscripts differ (P < 0.05).
      219
      Means within a row with no common superscripts differ (P < 0.05).
      184
      Means within a row with no common superscripts differ (P < 0.05).
      176
      Means within a row with no common superscripts differ (P < 0.05).
      10.9<0.02
       BC-Group 2
      BC-Group 2 = Ile, Leu, Val.
      78
      Means within a row with no common superscripts differ (P < 0.05).
      100
      Means within a row with no common superscripts differ (P < 0.05).
      161
      Means within a row with no common superscripts differ (P < 0.05).
      124
      Means within a row with no common superscripts differ (P < 0.05).
      89
      Means within a row with no common superscripts differ (P < 0.05).
      8.0<0.01
       NB-Group 2
      NB-Group 2 = Arg, Lys, Thr.
      61.3
      Means within a row with no common superscripts differ (P < 0.05).
      73.9
      Means within a row with no common superscripts differ (P < 0.05).
      58.3
      Means within a row with no common superscripts differ (P < 0.05).
      59.6
      Means within a row with no common superscripts differ (P < 0.05).
      86.9
      Means within a row with no common superscripts differ (P < 0.05).
      4.20<0.01
       NEAA
      NEAA = Ala, Asn, Asp, Cit, Cys, Gln, Glu, Gly, Orn, Pro, Ser, Tyr.
      14512411611913613.50.53
       TAA
      TAA = EAA + NEAA.
      31333336533934622.60.57
       Arg18.921.520.119.723.51.080.10
       His7.28.57.48.48.60.570.13
       Ile20.4
      Means within a row with no common superscripts differ (P < 0.05).
      27.2
      Means within a row with no common superscripts differ (P < 0.05).
      39.9
      Means within a row with no common superscripts differ (P < 0.05).
      33.6
      Means within a row with no common superscripts differ (P < 0.05).
      23.6
      Means within a row with no common superscripts differ (P < 0.05).
      2.03<0.01
       Leu30.1
      Means within a row with no common superscripts differ (P < 0.05).
      40.9
      Means within a row with no common superscripts differ (P < 0.05).
      60.6
      Means within a row with no common superscripts differ (P < 0.05).
      48.8
      Means within a row with no common superscripts differ (P < 0.05).
      35.0
      Means within a row with no common superscripts differ (P < 0.05).
      2.49<0.01
       Lys26.2
      Means within a row with no common superscripts differ (P < 0.05).
      34.1
      Means within a row with no common superscripts differ (P < 0.05).
      22.3
      Means within a row with no common superscripts differ (P < 0.05).
      23.2
      Means within a row with no common superscripts differ (P < 0.05).
      42.8
      Means within a row with no common superscripts differ (P < 0.05).
      2.81<0.01
       Met6.98.36.87.97.80.450.03
       Phe12.0
      Means within a row with no common superscripts differ (P < 0.05).
      14.9
      Means within a row with no common superscripts differ (P < 0.05).
      11.9
      Means within a row with no common superscripts differ (P < 0.05).
      15.8
      Means within a row with no common superscripts differ (P < 0.05).
      14.2
      Means within a row with no common superscripts differ (P < 0.05).
      0.74<0.01
       Thr16.2
      Means within a row with no common superscripts differ (P < 0.05).
      18.4
      Means within a row with no common superscripts differ (P < 0.05).
      15.8
      Means within a row with no common superscripts differ (P < 0.05).
      16.7
      Means within a row with no common superscripts differ (P < 0.05).
      20.5
      Means within a row with no common superscripts differ (P < 0.05).
      1.060.02
       Trp2.52.93.03.22.90.250.54
       Val27.0
      Means within a row with no common superscripts differ (P < 0.05).
      32.1
      Means within a row with no common superscripts differ (P < 0.05).
      60.4
      Means within a row with no common superscripts differ (P < 0.05).
      42.0
      Means within a row with no common superscripts differ (P < 0.05).
      30.2
      Means within a row with no common superscripts differ (P < 0.05).
      3.78<0.01
       Ala25.1
      Means within a row with no common superscripts differ (P < 0.05).
      6.4
      Means within a row with no common superscripts differ (P < 0.05).
      12.8
      Means within a row with no common superscripts differ (P < 0.05).
      4.8
      Means within a row with no common superscripts differ (P < 0.05).
      7.5
      Means within a row with no common superscripts differ (P < 0.05).
      4.320.03
       Asn7.0
      Means within a row with no common superscripts differ (P < 0.05).
      8.7
      Means within a row with no common superscripts differ (P < 0.05).
      5.3
      Means within a row with no common superscripts differ (P < 0.05).
      7.7
      Means within a row with no common superscripts differ (P < 0.05).
      8.2
      Means within a row with no common superscripts differ (P < 0.05).
      0.650.01
       Asp1.20.10.80.91.50.300.06
       Cit1.3
      Means within a row with no common superscripts differ (P < 0.05).
      0.2
      Means within a row with no common superscripts differ (P < 0.05).
      2.8
      Means within a row with no common superscripts differ (P < 0.05).
      1.7
      Means within a row with no common superscripts differ (P < 0.05).
      1.2
      Means within a row with no common superscripts differ (P < 0.05).
      0.550.07
       Cys0.12
      Means within a row with no common superscripts differ (P < 0.05).
      0.34
      Means within a row with no common superscripts differ (P < 0.05).
      0.18
      Means within a row with no common superscripts differ (P < 0.05).
      0.54
      Means within a row with no common superscripts differ (P < 0.05).
      0.25
      Means within a row with no common superscripts differ (P < 0.05).
      0.0830.03
       Gln40.541.430.844.444.85.220.36
       Glu21.416.620.115.721.92.640.23
       Gly13.19.311.68.19.83.070.78
       Orn10.110.010.49.114.51.500.14
       Pro8.0
      Means within a row with no common superscripts differ (P < 0.05).
      5.6
      Means within a row with no common superscripts differ (P < 0.05).
      6.2
      Means within a row with no common superscripts differ (P < 0.05).
      5.2
      Means within a row with no common superscripts differ (P < 0.05).
      6.1
      Means within a row with no common superscripts differ (P < 0.05).
      0.510.02
       Ser7.313.55.010.46.93.150.16
       Tyr10.011.710.210.911.40.700.32
      a–d Means within a row with no common superscripts differ (P < 0.05).
      1 Abomasal infusion treatments were saline (negative control) and 4 different AA profiles. SAL = 0.9% saline infusion; EAAC = a complete mixture of EAA (Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, and Val); ILV = mixture of Ile, Leu, and Val; GR1+ILV = mixture of His, Ile, Leu, Met, Phe, Trp, and Val; GR1+ALT = mixture of Arg, His, Lys, Met, Phe, Thr, and Trp. Amino acids within treatments were infused in amounts relative to their content in casein. All AA treatments supplied 562 g of MP/d, and solutions were delivered in 15-L batches.
      2 EAA = Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Val.
      3 Group 1 = His, Met, Phe+Tyr, Trp.
      4 Group 2 = Arg, Ile, Leu, Lys, Thr, Val.
      5 BC-Group 2 = Ile, Leu, Val.
      6 NB-Group 2 = Arg, Lys, Thr.
      7 NEAA = Ala, Asn, Asp, Cit, Cys, Gln, Glu, Gly, Orn, Pro, Ser, Tyr.
      8 TAA = EAA + NEAA.

      Mammary Clearance of AA Groups

      Mammary clearance of EAA decreased in response to all AA infusions compared with SAL (P ≤ 0.02; Table 6). Infusion of EAAC decreased clearance of group 2 AA (P ≤ 0.03) compared with SAL. Infusion of ILV decreased clearance of TAA, group 2 AA, and BC-group 2 AA (P ≤ 0.02) compared with SAL. Infusion of ILV increased clearance of group 1 AA (P < 0.01) over all other AA infusions, and increased clearance of NB-group 2 AA over EAAC and GR1+ALT (P ≤ 0.01). Infusion of GR1+ILV decreased mammary clearance of group 1 and group 2 AA (P ≤ 0.02) and increased clearance of NB-group 2 AA (P = 0.05) compared with SAL. Infusion of GR1+ILV increased clearance of NB-group 2 AA over EAAC (P = 0.02). Compared with SAL, infusion of GR1+ALT decreased mammary clearance of group 1 AA and NB-group 2 AA (P ≤ 0.05). Infusion of GR1+ALT increased mammary clearance of BC-group 2 AA over all other AA infusions (P = 0.01). Clearance of NB-group 2 AA increased over GR1+ALT with GR1+ILV infusion (P = 0.01).
      Table 6Mammary clearances (L/h) of AA groups in lactating dairy cows receiving 5-d abomasal infusions of saline (SAL) or 562 g/d of AA as a complete mixture of EAA (EAAC), as a mixture of Ile, Leu, and Val (ILV), as a mixture of group 1 AA and Ile, Leu, and Val (GR1+ILV), and as a mixture of group 1 AA and Arg, Lys, and Thr (GR1+ALT)
      ItemTreatment
      Abomasal infusion treatments were saline (negative control) and 4 different AA profiles. SAL = 0.9% saline infusion; EAAC = a complete mixture of EAA (Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, and Val); ILV = mixture of Ile, Leu, and Val; GR1+ILV = mixture of His, Ile, Leu, Met, Phe, Trp, and Val; GR1+ALT = mixture of Arg, His, Lys, Met, Phe, Thr, and Trp. Amino acids within treatments were infused in amounts relative to their content in casein. All AA treatments supplied 562 g of MP/d, and solutions were delivered in 15-L batches.
      SEMP-value
      SALEAACILVGR1+ILVGR1+ALT
      EAA
      EAA = Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Val.
      400
      Means within a row with no common superscripts differ (P < 0.05).
      215
      Means within a row with no common superscripts differ (P < 0.05).
      145
      Means within a row with no common superscripts differ (P < 0.05).
      190
      Means within a row with no common superscripts differ (P < 0.05).
      226
      Means within a row with no common superscripts differ (P < 0.05).
      30.1<0.01
      Group 1
      Group 1 = His, Met, Phe+Tyr, Trp.
      409
      Means within a row with no common superscripts differ (P < 0.05).
      237
      Means within a row with no common superscripts differ (P < 0.05).
      575
      Means within a row with no common superscripts differ (P < 0.05).
      127
      Means within a row with no common superscripts differ (P < 0.05).
      142
      Means within a row with no common superscripts differ (P < 0.05).
      54.0<0.01
      Group 2
      Group 2 = Arg, Ile, Leu, Lys, Thr, Val.
      421
      Means within a row with no common superscripts differ (P < 0.05).
      225
      Means within a row with no common superscripts differ (P < 0.05).
      134
      Means within a row with no common superscripts differ (P < 0.05).
      222
      Means within a row with no common superscripts differ (P < 0.05).
      287
      Means within a row with no common superscripts differ (P < 0.05).
      40.5<0.01
      BC-Group 2
      BC-Group 2 = Ile, Leu, Val.
      355
      Means within a row with no common superscripts differ (P < 0.05).
      190
      Means within a row with no common superscripts differ (P < 0.05).
      105
      Means within a row with no common superscripts differ (P < 0.05).
      166
      Means within a row with no common superscripts differ (P < 0.05).
      475
      Means within a row with no common superscripts differ (P < 0.05).
      46.3<0.01
      NB-Group 2
      NB-Group 2 = Arg, Lys, Thr.
      566
      Means within a row with no common superscripts differ (P < 0.05).
      312
      Means within a row with no common superscripts differ (P < 0.05).
      689
      Means within a row with no common superscripts differ (P < 0.05).
      863
      Means within a row with no common superscripts differ (P < 0.05).
      249
      Means within a row with no common superscripts differ (P < 0.05).
      65.8<0.01
      NEAA
      NEAA = Ala, Asn, Asp, Cit, Cys, Gln, Glu, Gly, Orn, Pro, Ser, Tyr.
      15516312412513416.50.32
      TAA
      TAA = EAA + NEAA.
      227
      Means within a row with no common superscripts differ (P < 0.05).
      190
      Means within a row with no common superscripts differ (P < 0.05).
      136
      Means within a row with no common superscripts differ (P < 0.05).
      157
      Means within a row with no common superscripts differ (P < 0.05).
      180
      Means within a row with no common superscripts differ (P < 0.05).
      16.80.02
      a–d Means within a row with no common superscripts differ (P < 0.05).
      1 Abomasal infusion treatments were saline (negative control) and 4 different AA profiles. SAL = 0.9% saline infusion; EAAC = a complete mixture of EAA (Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, and Val); ILV = mixture of Ile, Leu, and Val; GR1+ILV = mixture of His, Ile, Leu, Met, Phe, Trp, and Val; GR1+ALT = mixture of Arg, His, Lys, Met, Phe, Thr, and Trp. Amino acids within treatments were infused in amounts relative to their content in casein. All AA treatments supplied 562 g of MP/d, and solutions were delivered in 15-L batches.
      2 EAA = Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Val.
      3 Group 1 = His, Met, Phe+Tyr, Trp.
      4 Group 2 = Arg, Ile, Leu, Lys, Thr, Val.
      5 BC-Group 2 = Ile, Leu, Val.
      6 NB-Group 2 = Arg, Lys, Thr.
      7 NEAA = Ala, Asn, Asp, Cit, Cys, Gln, Glu, Gly, Orn, Pro, Ser, Tyr.
      8 TAA = EAA + NEAA.

      Mammary Gland AA U:O

      Infusion of EAAC did not affect the mammary gland U:O of any EAA group or individual EAA compared with SAL (P > 0.95; Table 7). Infusion of ILV increased U:O of EAA, group 2, and BC-group 2 AA (as a group and individually) over SAL and all other AA infusions (P < 0.01). Individually, U:O of Ile and Leu increased and U:O of Thr decreased with GR1+ILV compared with SAL (P ≤ 0.05), U:O of Arg decreased with GR1+ILV compared with ILV and GR1+ALT (P ≤ 0.02), and U:O of Lys decreased with GR1+ILV compared with EAAC (P = 0.03). Infusion of GR1+ALT increased U:O of NB-group 2 AA and individual U:O of Lys over SAL and all AA infusions (P ≤ 0.05), and increased U:O of Thr over EAAC and GR1+ILV (P ≤ 0.04).
      Table 7Mammary gland AA uptake to milk output ratios in lactating dairy cows receiving 5-d abomasal infusions of saline (SAL) or 562 g/d of AA as a complete mixture of EAA (EAAC), as a mixture of Ile, Leu, and Val (ILV), as a mixture of group 1 AA and Ile, Leu, and Val (GR1+ILV), and as a mixture of group 1 AA and Arg, Lys, and Thr (GR1+ALT)
      ItemTreatment
      Abomasal infusion treatments were saline (negative control) and 4 different AA profiles. SAL = 0.9% saline infusion; EAAC = a complete mixture of EAA (Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, and Val); ILV = mixture of Ile, Leu, and Val; GR1+ILV = mixture of His, Ile, Leu, Met, Phe, Trp, and Val; GR1+ALT = mixture of Arg, His, Lys, Met, Phe, Thr, and Trp. Amino acids within treatments were infused in amounts relative to their content in casein. All AA treatments supplied 562 g of MP/d, and solutions were delivered in 15-L batches.
      SEMP-value
      SALEAACILVGR1+ILVGR1+ALT
      EAA
      EAA = Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Val.
      1.23
      Means within a row with no common superscripts differ (P < 0.05).
      1.28
      Means within a row with no common superscripts differ (P < 0.05).
      1.84
      Means within a row with no common superscripts differ (P < 0.05).
      1.35
      Means within a row with no common superscripts differ (P < 0.05).
      1.35
      Means within a row with no common superscripts differ (P < 0.05).
      0.078<0.01
      Group 1
      Group 1 = His, Met, Phe+Tyr, Trp.
      1.01
      Value does not differ from 1.00 (P > 0.10).
      1.00
      Value does not differ from 1.00 (P > 0.10).
      1.01
      Value does not differ from 1.00 (P > 0.10).
      1.00
      Value does not differ from 1.00 (P > 0.10).
      1.01
      Value does not differ from 1.00 (P > 0.10).
      0.0130.83
      Group 2
      Group 2 = Arg, Ile, Leu, Lys, Thr, Val.
      1.28
      Means within a row with no common superscripts differ (P < 0.05).
      1.34
      Means within a row with no common superscripts differ (P < 0.05).
      2.04
      Means within a row with no common superscripts differ (P < 0.05).
      1.42
      Means within a row with no common superscripts differ (P < 0.05).
      1.43
      Means within a row with no common superscripts differ (P < 0.05).
      0.097<0.01
      BC-Group 2
      BC-Group 2 = Ile, Leu, Val.
      1.20
      Means within a row with no common superscripts differ (P < 0.05).
      Value does not differ from 1.00 (P > 0.10).
      1.29
      Means within a row with no common superscripts differ (P < 0.05).
      2.52
      Means within a row with no common superscripts differ (P < 0.05).
      1.61
      Means within a row with no common superscripts differ (P < 0.05).
      1.23
      Means within a row with no common superscripts differ (P < 0.05).
      Value does not differ from 1.00 (P > 0.10).
      0.138<0.01
      NB-Group 2
      NB-Group 2 = Arg, Lys, Thr.
      1.40
      Means within a row with no common superscripts differ (P < 0.05).
      1.41
      Means within a row with no common superscripts differ (P < 0.05).
      1.32
      Means within a row with no common superscripts differ (P < 0.05).
      1.13
      Means within a row with no common superscripts differ (P < 0.05).
      †Value does not differ from 1.00 (tendency; 0.10 ≥ P > 0.05); if no symbol, the value differs from 1.00 (P < 0.05).
      1.72
      Means within a row with no common superscripts differ (P < 0.05).
      0.066<0.01
      NEAA
      NEAA = Ala, Asn, Asp, Cit, Cys, Gln, Glu, Gly, Orn, Pro, Ser, Tyr.
      0.80
      Means within a row with no common superscripts differ (P < 0.05).
      0.55
      Means within a row with no common superscripts differ (P < 0.05).
      0.58
      Means within a row with no common superscripts differ (P < 0.05).
      0.53
      Means within a row with no common superscripts differ (P < 0.05).
      0.62
      Means within a row with no common superscripts differ (P < 0.05).
      0.0550.03
      TAA
      TAA = EAA + NEAA.
      0.98
      Means within a row with no common superscripts differ (P < 0.05).
      Value does not differ from 1.00 (P > 0.10).
      0.86
      Means within a row with no common superscripts differ (P < 0.05).
      1.11
      Means within a row with no common superscripts differ (P < 0.05).
      0.88
      Means within a row with no common superscripts differ (P < 0.05).
      0.91
      Means within a row with no common superscripts differ (P < 0.05).
      Value does not differ from 1.00 (P > 0.10).
      0.0440.01
      TAA-N
      TAA on a N basis.
      1.01
      Means within a row with no common superscripts differ (P < 0.05).
      Value does not differ from 1.00 (P > 0.10).
      0.91
      Means within a row with no common superscripts differ (P < 0.05).
      †Value does not differ from 1.00 (tendency; 0.10 ≥ P > 0.05); if no symbol, the value differs from 1.00 (P < 0.05).
      1.20
      Means within a row with no common superscripts differ (P < 0.05).
      0.93
      Means within a row with no common superscripts differ (P < 0.05).
      Value does not differ from 1.00 (P > 0.10).
      0.96
      Means within a row with no common superscripts differ (P < 0.05).
      Value does not differ from 1.00 (P > 0.10).
      0.045<0.01
      Arg2.55
      Means within a row with no common superscripts differ (P < 0.05).
      2.40
      Means within a row with no common superscripts differ (P < 0.05).
      2.69
      Means within a row with no common superscripts differ (P < 0.05).
      2.20
      Means within a row with no common superscripts differ (P < 0.05).
      2.68
      Means within a row with no common superscripts differ (P < 0.05).
      0.0940.01
      His1.101.07
      Value does not differ from 1.00 (P > 0.10).
      1.101.07
      Value does not differ from 1.00 (P > 0.10).
      1.140.0430.74
      Ile1.25
      Means within a row with no common superscripts differ (P < 0.05).
      Value does not differ from 1.00 (P > 0.10).
      1.40
      Means within a row with no common superscripts differ (P < 0.05).
      2.48
      Means within a row with no common superscripts differ (P < 0.05).
      1.72
      Means within a row with no common superscripts differ (P < 0.05).
      1.29
      Means within a row with no common superscripts differ (P < 0.05).
      †Value does not differ from 1.00 (tendency; 0.10 ≥ P > 0.05); if no symbol, the value differs from 1.00 (P < 0.05).
      0.147<0.01
      Leu1.09
      Means within a row with no common superscripts differ (P < 0.05).
      Value does not differ from 1.00 (P > 0.10).
      1.24
      Means within a row with no common superscripts differ (P < 0.05).
      2.24
      Means within a row with no common superscripts differ (P < 0.05).
      1.48
      Means within a row with no common superscripts differ (P < 0.05).
      1.12
      Means within a row with no common superscripts differ (P < 0.05).
      Value does not differ from 1.00 (P > 0.10).
      0.112<0.01
      Lys1.22
      Means within a row with no common superscripts differ (P < 0.05).
      †Value does not differ from 1.00 (tendency; 0.10 ≥ P > 0.05); if no symbol, the value differs from 1.00 (P < 0.05).
      1.33
      Means within a row with no common superscripts differ (P < 0.05).
      1.04
      Means within a row with no common superscripts differ (P < 0.05).
      Value does not differ from 1.00 (P > 0.10).
      0.90
      Means within a row with no common superscripts differ (P < 0.05).
      Value does not differ from 1.00 (P > 0.10).
      1.75
      Means within a row with no common superscripts differ (P < 0.05).
      0.111<0.01
      Met1.00
      Value does not differ from 1.00 (P > 0.10).
      1.00
      Value does not differ from 1.00 (P > 0.10).
      0.98
      Value does not differ from 1.00 (P > 0.10).
      0.96
      Value does not differ from 1.00 (P > 0.10).
      0.98
      Value does not differ from 1.00 (P > 0.10).
      0.0290.67
      Phe1.101.131.081.191.150.0340.16
      Thr1.09
      Means within a row with no common superscripts differ (P < 0.05).
      1.02
      Means within a row with no common superscripts differ (P < 0.05).
      Value does not differ from 1.00 (P > 0.10).
      1.04
      Means within a row with no common superscripts differ (P < 0.05).
      Value does not differ from 1.00 (P > 0.10).
      0.93
      Means within a row with no common superscripts differ (P < 0.05).
      †Value does not differ from 1.00 (tendency; 0.10 ≥ P > 0.05); if no symbol, the value differs from 1.00 (P < 0.05).
      1.19
      Means within a row with no common superscripts differ (P < 0.05).
      0.033<0.01
      Trp0.89
      Value does not differ from 1.00 (P > 0.10).
      0.841.00
      Value does not differ from 1.00 (P > 0.10).
      0.91
      Value does not differ from 1.00 (P > 0.10).
      0.840.0640.42
      Val1.29
      Means within a row with no common superscripts differ (P < 0.05).
      Value does not differ from 1.00 (P > 0.10).
      1.29
      Means within a row with no common superscripts differ (P < 0.05).
      Value does not differ from 1.00 (P > 0.10).
      2.91
      Means within a row with no common superscripts differ (P < 0.05).
      1.68
      Means within a row with no common superscripts differ (P < 0.05).
      1.32
      Means within a row with no common superscripts differ (P < 0.05).
      Value does not differ from 1.00 (P > 0.10).
      0.182<0.01
      Ala1.87
      Means within a row with no common superscripts differ (P < 0.05).
      0.35
      Means within a row with no common superscripts differ (P < 0.05).
      0.97
      Means within a row with no common superscripts differ (P < 0.05).
      Value does not differ from 1.00 (P > 0.10).
      0.26
      Means within a row with no common superscripts differ (P < 0.05).
      0.41
      Means within a row with no common superscripts differ (P < 0.05).
      †Value does not differ from 1.00 (tendency; 0.10 ≥ P > 0.05); if no symbol, the value differs from 1.00 (P < 0.05).
      0.3030.01
      Asn0.590.620.440.550.610.0430.06
      Asp0.07
      Means within a row with no common superscripts differ (P < 0.05).
      0.01
      Means within a row with no common superscripts differ (P < 0.05).
      0.05
      Means within a row with no common superscripts differ (P < 0.05).
      0.05
      Means within a row with no common superscripts differ (P < 0.05).
      0.08
      Means within a row with no common superscripts differ (P < 0.05).
      0.0160.04
      Cys0.05
      Means within a row with no common superscripts differ (P < 0.05).
      0.11
      Means within a row with no common superscripts differ (P < 0.05).
      0.06
      Means within a row with no common superscripts differ (P < 0.05).
      0.16
      Means within a row with no common superscripts differ (P < 0.05).
      0.08
      Means within a row with no common superscripts differ (P < 0.05).
      0.0230.03
      Gln1.701.421.25
      Value does not differ from 1.00 (P > 0.10).
      1.531.580.1820.48
      Glu0.520.330.480.310.470.0610.10
      Gly1.49
      Value does not differ from 1.00 (P > 0.10).
      0.82
      Value does not differ from 1.00 (P > 0.10).
      1.21
      Value does not differ from 1.00 (P > 0.10).
      0.71
      Value does not differ from 1.00 (P > 0.10).
      0.90
      Value does not differ from 1.00 (P > 0.10).
      0.2990.37
      Pro0.25
      Means within a row with no common superscripts differ (P < 0.05).
      0.14
      Means within a row with no common superscripts differ (P < 0.05).
      0.19
      Means within a row with no common superscripts differ (P < 0.05).
      0.13
      Means within a row with no common superscripts differ (P < 0.05).
      0.15
      Means within a row with no common superscripts differ (P < 0.05).
      0.017<0.01
      Ser0.360.510.190.400.260.1260.23
      Tyr0.900.870.920.810.890.0330.16
      a–c Means within a row with no common superscripts differ (P < 0.05).
      1 Abomasal infusion treatments were saline (negative control) and 4 different AA profiles. SAL = 0.9% saline infusion; EAAC = a complete mixture of EAA (Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, and Val); ILV = mixture of Ile, Leu, and Val; GR1+ILV = mixture of His, Ile, Leu, Met, Phe, Trp, and Val; GR1+ALT = mixture of Arg, His, Lys, Met, Phe, Thr, and Trp. Amino acids within treatments were infused in amounts relative to their content in casein. All AA treatments supplied 562 g of MP/d, and solutions were delivered in 15-L batches.
      2 EAA = Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Val.
      3 Group 1 = His, Met, Phe+Tyr, Trp.
      4 Group 2 = Arg, Ile, Leu, Lys, Thr, Val.
      5 BC-Group 2 = Ile, Leu, Val.
      6 NB-Group 2 = Arg, Lys, Thr.
      7 NEAA = Ala, Asn, Asp, Cit, Cys, Gln, Glu, Gly, Orn, Pro, Ser, Tyr.
      8 TAA = EAA + NEAA.
      9 TAA on a N basis.
      * Value does not differ from 1.00 (P > 0.10).
      †Value does not differ from 1.00 (tendency; 0.10 ≥ P > 0.05); if no symbol, the value differs from 1.00 (P < 0.05).
      Infusion of EAAC decreased U:O of NEAA and individual U:O of Ala, Asp, and Pro compared with SAL (P ≤ 0.05). Compared with SAL, infusion of GR1+ILV decreased U:O of NEAA and individual U:O of Ala and Pro (P ≤ 0.03), and increased U:O of Cys (P = 0.02). Infusion of GR1+ALT decreased individual U:O of Ala and Pro compared with SAL (P ≤ 0.05). Infusion of ILV increased U:O of TAA over EAAC and GR1+ILV (P ≤ 0.02) and increased TAA U:O expressed on an N basis over all other AA infusions (P ≤ 0.03). Total AA U:O expressed on an N basis was greater than 1 during ILV infusion, tended to differ from 1 during EAAC infusion, but did not differ from 1 on all other treatments (P ≤ 0.10).

      Glucose Metabolism and 13CO2 Production

      Arterial plasma glucose concentration, arterial glucose IE, and mammary net uptake of glucose were not affected by AA infusions (P ≥ 0.11; Table 8). Infusion of GR1+ALT increased glucose WB Ra over SAL and ILV (P = 0.05). The calculated glucose required for lactose and fat synthesis was not affected by treatment (P ≥ 0.23), and mammary glucose uptake was sufficient to cover these estimated requirements (i.e., glucose balance across treatments did not differ from zero; P ≥ 0.10). Neither lactose output as a proportion of mammary glucose uptake nor as a proportion of WB Ra was affected by treatment (P ≥ 0.39), averaging 0.67 and 0.53, respectively. The proportion of mammary glucose uptake relative to WB Ra was not affected by treatment (P = 0.89) and averaged 0.76. The IE of glucose and galactose in milk lactose and the ratio of galactose IE relative to glucose IE (average 0.72) was not affected by treatment (P ≥ 0.74). Isotopic enrichment of glucose in milk lactose was higher than IE of galactose (P < 0.01). Total 13CO2 production (Figure 1) between 0500 h on d 5 and 0500 h on d 6 did not differ between treatments (P = 0.57), and averaged 31.4, 28.8, 32.5, 33.6, and 27.0 mmol for SAL, EAAC, ILV, GR1+ILV, and GR1+ALT, respectively, with an average SEM of 3.61. Whole-body glucose oxidation and the proportion of WB glucose oxidation relative to WB Ra were not affected by treatment (P ≥ 0.45) and averaged 118 mmol/h and 0.17, respectively, across treatments.
      Table 8Whole-body and mammary glucose metabolism in lactating dairy cows receiving 5-d abomasal infusions of saline (SAL) or 562 g/d of AA as a complete mixture of EAA (EAAC), as a mixture of Ile, Leu, and Val (ILV), as a mixture of group 1 AA and Ile, Leu, and Val (GR1+ILV), and as a mixture of group 1 AA and Arg, Lys, and Thr (GR1+ALT)
      ItemTreatment
      Abomasal infusion treatments were saline (negative control) and 4 different AA profiles. SAL = 0.9% saline infusion; EAAC = a complete mixture of EAA (Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, and Val); ILV = mixture of Ile, Leu, and Val; GR1+ILV = mixture of His, Ile, Leu, Met, Phe, Trp, and Val; GR1+ALT = mixture of Arg, His, Lys, Met, Phe, Thr, and Trp. Amino acids within treatments were infused in amounts relative to their content in casein. All AA treatments supplied 562 g of MP/d, and solutions were delivered in 15-L batches.
      SEMP-value
      SALEAACILVGR1+ILVGR1+ALT
      Arterial glucose, mM3.643.643.583.363.710.1400.49
      Arterial glucose IE, APE
      IE = isotopic enrichment; APE = atom percent excess.
      0.5280.5070.5250.4720.4590.02610.11
      WB Ra,
      WB = whole body; Ra = rate of glucose appearance.
      mmol/h
      651
      Means within a row with no common superscripts differ (P < 0.05).
      700
      Means within a row with no common superscripts differ (P < 0.05).
      658
      Means within a row with no common superscripts differ (P < 0.05).
      728
      Means within a row with no common superscripts differ (P < 0.05).
      757
      Means within a row with no common superscripts differ (P < 0.05).
      33.90.04
      Mammary glucose uptake, mmol/h55354952851060581.00.94
      Glucose required for lactose,
      Based on milk lactose and fat yield from the afternoon milking on d 4. Requirements for lactose and fat estimated based on calculations of Dijkstra et al. (1996).
      mmol/h
      35939237239739123.50.25
      Glucose required for fat,
      Based on milk lactose and fat yield from the afternoon milking on d 4. Requirements for lactose and fat estimated based on calculations of Dijkstra et al. (1996).
      mmol/h
      101991071151047.00.23
      Mammary glucose balance,
      Glucose balance = glucose uptake – glucose required for lactose – glucose required for fat. Values do not differ from zero (P > 0.10).
      mmol/h
      945948−29470.80.86
      Lactose output
      Based on milk lactose yield from the afternoon milking on d 4.
      :mammary glucose uptake
      0.640.730.600.740.620.0840.39
      Mammary glucose uptake:WB Ra0.820.750.780.700.760.1220.89
      Lactose output
      Based on milk lactose yield from the afternoon milking on d 5.
      :WB Ra
      0.510.570.540.520.510.0320.73
      WB glucose oxidation, mmol/h12411311310513411.70.55
      WB glucose oxidation:WB Ra0.190.160.180.150.180.0180.45
      Lactose IE
       Glucose,
      Difference between glucose APE and galactose APE differs from zero (P < 0.01).
      APE
      0.1490.1340.1340.1260.1210.01670.74
       Galactose, APE0.1080.1000.0960.0890.0880.01350.81
       Galactose IE/glucose IE0.710.730.710.700.730.0250.83
      a,b Means within a row with no common superscripts differ (P < 0.05).
      1 Abomasal infusion treatments were saline (negative control) and 4 different AA profiles. SAL = 0.9% saline infusion; EAAC = a complete mixture of EAA (Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, and Val); ILV = mixture of Ile, Leu, and Val; GR1+ILV = mixture of His, Ile, Leu, Met, Phe, Trp, and Val; GR1+ALT = mixture of Arg, His, Lys, Met, Phe, Thr, and Trp. Amino acids within treatments were infused in amounts relative to their content in casein. All AA treatments supplied 562 g of MP/d, and solutions were delivered in 15-L batches.
      2 IE = isotopic enrichment; APE = atom percent excess.
      3 WB = whole body; Ra = rate of glucose appearance.
      4 Based on milk lactose and fat yield from the afternoon milking on d 4. Requirements for lactose and fat estimated based on calculations of
      • Dijkstra J.
      • France J.
      • Assis A.G.
      • Neal H. D. St. C.
      • Campos O.F.
      • Aroeira L.M.J.
      Simulation of digestion in cattle fed sugarcane: Prediction of nutrient supply for milk production with locally available supplements.
      .
      5 Glucose balance = glucose uptake – glucose required for lactose – glucose required for fat. Values do not differ from zero (P > 0.10).
      6 Based on milk lactose yield from the afternoon milking on d 4.
      7 Based on milk lactose yield from the afternoon milking on d 5.
      8 Difference between glucose APE and galactose APE differs from zero (P < 0.01).

      DISCUSSION

      The current results complement previously reported energy and N balance from the same study (
      • Nichols K.
      • Bannink A.
      • Dijkstra J.
      Energy and nitrogen balance of dairy cattle as affected by provision of different essential amino acid profiles at the same metabolizable protein supply.
      ). Examinations of the effects of incomplete EAA profiles on mammary gland metabolism have been conducted under conditions where single AA or groups of AA are subtracted from 5 to 14 d postruminal infusions of complete AA profiles (
      • Bequette B.J.
      • Hanigan M.D.
      • Calder A.G.
      • Reynolds C.K.
      • Lobley G.E.
      • MacRae J.C.
      Amino acid exchange by the mammary gland of lactating goats when histidine limits milk production.
      ;
      • Doepel L.
      • Lapierre H.
      Deletion of arginine from an abomasal infusion of amino acids does not decrease milk protein yield in Holstein cows.
      ;
      • Doepel L.
      • Hewage I.I.
      • Lapierre H.
      Milk protein yield and mammary metabolism are affected by phenylalanine deficiency but not by threonine or tryptophan deficiency.
      ). However, the reduced MP supply from the subtracted AA in these studies is not compensated. Therefore, the first objective of the present work was to examine mammary gland responses to incomplete EAA profiles at a constant supplemental MP level for 5 d, with the general hypothesis that intramammary metabolism of the supplemented EAA in the incomplete profiles would increase in support of milk protein synthesis.

      Mammary Plasma Flow

      Mammary plasma flow was fastest during ILV infusion, increasing 38% over that on EAAC and GR1+ILV. Mammary plasma flow can be altered during excessive or deficient supply of milk precursors in an attempt to maintain extracellular and intracellular concentrations of precursor substrates required for milk component synthesis, particularly when single EAA, acetate, or BHB are deficient in arterial circulation (
      • Cant J.P.
      • Berthiaume R.
      • Lapierre H.
      • Luimes P.H.
      • McBride B.W.
      • Pacheco D.
      Responses of the bovine mammary glands to absorptive supply of single amino acids.
      ). In previous studies, extra MP from EAA and NEAA (
      • Galindo C.E.
      • Ouellet D.R.
      • Pellerin D.
      • Lemosquet S.
      • Ortigues-Marty I.
      • Lapierre H.
      Effect of amino acid or casein supply on whole-body, splanchnic, and mammary glucose kinetics in lactating dairy cows.
      ;
      • Nichols K.
      • van Laar H.
      • Bannink A.
      • Dijkstra J.
      Mammary gland utilization of amino acids and energy metabolites differs when dairy cow rations are isoenergetically supplemented with protein and fat.
      ), or as a mixture of EAA only (
      • Nichols K.
      • Bannink A.
      • Doelman J.
      • Dijkstra J.
      Mammary gland metabolite utilization in response to exogenous glucose or long-chain fatty acids at low and high metabolizable protein levels.
      ), did not affect MPF compared with no extra MP, which is in line with the result of the present experiment. However, the effects of EAA deletions on MPF are variable. In dairy cattle, removal of Lys from an abomasally infused complete AA profile infused for 7 d did not affect MPF (
      • Lapierre H.
      • Doepel L.
      • Milne E.
      • Lobley G.E.
      Responses in mammary and splanchnic metabolism to altered lysine supply in dairy cows.
      ), whereas removal of Thr from a complete AA profile infused for 10 d increased MPF by 50% (
      • Doepel L.
      • Hewage I.I.
      • Lapierre H.
      Milk protein yield and mammary metabolism are affected by phenylalanine deficiency but not by threonine or tryptophan deficiency.
      ). Deletion of Arg from a complete EAA profile infused for 14 d did not affect MPF compared with the complete profile, but decreased it 17% relative to control levels (
      • Doepel L.
      • Lapierre H.
      Deletion of arginine from an abomasal infusion of amino acids does not decrease milk protein yield in Holstein cows.
      ).
      • Haque M.N.
      • Guinard-Flament J.
      • Lamberton P.
      • Mustière C.
      • Lemosquet S.
      Changes in mammary metabolism in response to the provision of an ideal amino acid profile at 2 levels of metabolizable protein supply in dairy cows: Consequences on efficiency.
      postruminally infused an EAA profile missing Thr and Trp and observed no effect on MPF in dairy cattle after 14 d, whereas
      • Bequette B.J.
      • Hanigan M.D.
      • Calder A.G.
      • Reynolds C.K.
      • Lobley G.E.
      • MacRae J.C.
      Amino acid exchange by the mammary gland of lactating goats when histidine limits milk production.
      reported a 33% increase in MPF in goats when His was removed from a 7-d abomasal infusion of AA in the profile of microbial protein. In the current study, if MPF was responding to the absence of any single EAA, it should have also increased during the other incomplete infusions (i.e., GR1+ILV and GR1+ALT). The magnitude of effect on MPF when single EAA are removed from complete supplements could relate to the degree of change in arterial concentration of the other EAA (
      • Cant J.P.
      • Trout D.R.
      • Qiao F.
      • McBride B.W.
      Milk composition responses to unilateral arterial infusion of complete and histidine-lacking amino acid mixtures to the mammary gland of cows.
      ). The ILV infusion was relatively more imbalanced than GR1+ILV and GR1+ALT with respect to a complete EAA profile, and this may explain why MPF was only increased in response to ILV. This difference in magnitude of imbalance can be seen in the response of circulating EAA concentrations to the incomplete infusions, where ILV increased the circulating concentration of only the BC-group 2 AA compared with SAL, and GR1+ILV and GR1+ALT increased the concentration of all EAA (numerical increase only for Ile with GR1+ILV infusion) in the infusate profile compared with SAL. Changes in blood flow are often associated with concomitant changes in arterial concentration of acetate and BHB, particularly during glucogenic infusions where the arterial concentrations of these metabolites decrease and MPF increases (
      • Raggio G.
      • Lemosquet S.
      • Lobley G.E.
      • Rulquin H.
      • Lapierre H.
      Effect of casein and propionate supply on mammary protein metabolism in lactating dairy cows.
      ;
      • Curtis R.V.
      • Kim J.J.M.
      • Doelman J.
      • Cant J.P.
      Maintenance of plasma branched-chain amino acid concentrations during glucose infusion directs essential amino acids to extra-mammary tissues in lactating dairy cows.
      ;
      • Nichols K.
      • Bannink A.
      • Doelman J.
      • Dijkstra J.
      Mammary gland metabolite utilization in response to exogenous glucose or long-chain fatty acids at low and high metabolizable protein levels.
      ). A link between AA supply and MPF has been hypothesized to lie with the associated effects on arterial energy metabolites when EAA supply is increased (
      • Cant J.P.
      • Berthiaume R.
      • Lapierre H.
      • Luimes P.H.
      • McBride B.W.
      • Pacheco D.
      Responses of the bovine mammary glands to absorptive supply of single amino acids.
      ,
      • Cant J.P.
      • Kim J.J.M.
      • Cieslar S.R.L.
      • Doelman J.
      Symposium review: Amino acid uptake by the mammary glands: Where does the control lie?.
      ). However, arterial BHB concentration was not affected by the infused AA profiles in the current study (Supplemental Table S2). This adds credence to the suggestion that the effect of ILV infusion on MPF is related to the relative AA imbalance, and may indicate that the mechanisms regulating MPF under conditions of AA imbalance can override those responsible for maintaining the ATP status of the gland.

      Intramammary Flexibility During ILV Infusion

      Arterial concentration of BC-group 2 AA during ILV infusion was increased over EAAC and GR1+ILV (treatments where Ile, Leu, and Val were also infused), but AVD of this AA group during ILV infusion did not differ from that on EAAC and GR1+ILV. The higher uptake of BC-group 2 AA can hence be attributed to the faster MPF during ILV infusion. The U:O of BC-group 2 AA increased with ILV over all infusions, to approximately double the level observed on SAL, EAAC, and GR1+ALT, and 1.6-times that on GR1+ILV, in agreement with our hypothesis that intramammary catabolism of BC-group 2 AA would be elevated when they were infused in the absence of other EAA. Consistent with previous research [e.g.,
      • Bequette B.J.
      • Backwell F.R.C.
      • MacRae J.C.
      • Lobley G.E.
      • Crompton L.A.
      • Metcalf J.A.
      • Sutton J.D.
      Effect of intravenous amino acid infusion on leucine oxidation across the mammary gland of the lactating goat.
      who reported oxidation of 11–23% of mammary Leu uptake], this observation shows that a high level of intramammary catabolism of Ile, Leu, and Val is possible by the lactating mammary gland, at least over a 5-d period. The mammary gland obtains substantial N and carbon for de novo NEAA synthesis from catabolism of BC-group 2 AA taken up in excess relative to their output in milk (
      • Lapierre H.
      • Lobley G.E.
      • Doepel L.
      • Raggio G.
      • Rulquin H.
      • Lemosquet S.
      Triennial Lactation Symposium: Mammary metabolism of amino acids in dairy cows.
      ). The U:O of total AA-N increased with ILV over the other AA infusions, driven by the increased U:O of BC-group 2 AA, and unlike all other treatments was significantly greater than 1. The substantial difference in mammary uptake of BC-group 2 AA and consequently total AA-N, relative to their output in milk, suggests that catabolic products of Ile, Leu, and Val likely contributed to other mammary pathways (lactose and fat synthesis, and oxidation) above what was used for intramammary NEAA synthesis. This increased intramammary AA catabolism supports the lowest milk N efficiency observed on this treatment (
      • Nichols K.
      • Bannink A.
      • Dijkstra J.
      Energy and nitrogen balance of dairy cattle as affected by provision of different essential amino acid profiles at the same metabolizable protein supply.
      ) and the observation that despite the lower DMI, milk protein, fat, and lactose production did not differ between ILV and SAL. Further, sequestration of BC-group 2 AA in the mammary gland through accumulation in constitutive protein, their release as peptides, or their output in milk as free AA or peptides would all contribute to the U:O of total AA-N of >1, and would indicate that the mammary gland can function as an important clearance organ for BC-group 2 AA.

      Intramammary Group 2 AA Flexibility During GR1+ILV and GR1+ALT Infusion

      In line with stoichiometric transfer of their uptake into milk protein, mammary net uptake of group 1 AA did not differ between EAAC, GR1+ILV, and GR1+ALT. Differences in uptake of the group 2 AA were found between the incomplete infusions, where BC-group 2 AA uptake tended to be higher on GR1+ILV compared with GR1+ALT, and NB-group 2 AA uptake was higher on GR1+ALT compared with GR1+ILV. Infusion of GR1+ILV increased clearance of NB-group 2 AA (as a group and individually; Supplemental Table S1) over EAAC. Infusion of GR1+ALT increased mammary clearance of BC-group 2 AA and individual clearances of Leu and Val (Supplemental Table S1) over all other AA infusions. Because their uptakes were not different from EAAC, higher clearance of these EAA groups is due to their relatively lower arterial concentrations on the respective incomplete EAA infusions, and reflects mammary affinity for their sequestration over 5 d when supplementation of the other EAA stimulated milk protein synthesis.
      We hypothesized that the same level of milk protein synthesis between EAAC, GR1+ILV (in the absence of Arg, Lys, and Thr), and GR1+ALT (in the absence of Ile, Leu, and Val) was maintained through intramammary compensation between the group 2 AA over the 5-d infusion period. Intramammary catabolism of NB-group 2 AA tended to decrease (and tended toward unity) with GR1+ILV compared with EAAC. The U:O of BC-group 2 AA numerically increased 25% over EAAC with GR1+ILV infusion. Therefore, intramammary catabolism of BC-group 2 AA could have compensated for lower levels of NB-group 2 AA during GR1+ILV infusion, in agreement with our hypothesis. During GR1+ALT infusion, intramammary catabolism of NB-group 2 AA increased over EAAC, but the U:O of BC-group 2 AA (individually and as a group) was not different from EAAC during GR1+ALT infusion. This difference in intramammary catabolism of the EAA groups absent from the respective infusions (i.e., NB-group 2 AA catabolism tended to decrease on GR1+ILV compared with EAAC, whereas BC-group 2 AA catabolism was not different from EAAC on GR1+ALT) arose primarily from the decrease in intramammary catabolism of Lys during GR1+ILV infusion. The U:O of Lys on GR1+ILV dropped from 1.33 on EAAC to 0.90 (not different from unity). When considered together with the U:O of Lys that was observed on ILV (1.04; not different from unity), our findings suggest that when intramammary BC-group 2 AA levels are high, Lys catabolism by the gland becomes minor or not obligate. Similarly,
      • Lapierre H.
      • Doepel L.
      • Milne E.
      • Lobley G.E.
      Responses in mammary and splanchnic metabolism to altered lysine supply in dairy cows.
      removed Lys from a complete AA mixture and observed reduced U:O of Lys accompanied by reduced Lys-N transfer into NEAA, whereas milk protein yield was not affected. In particular, abundant intramammary levels of BC-group 2 AA or Arg could have compensated for the reduced contribution of Lys to intramammary amino-N exchanges in support of milk protein synthesis during GR1+ILV and ILV infusion (
      • Bequette B.J.
      • Hanigan M.D.
      • Lapierre H.
      Mammary uptake and metabolism of amino acids by lactating ruminants.
      ). Intracellular requirements for Arg appear to hold a constant level of priority for intramammary catabolism across a range of digestive supplies (33 to 175 g/d;
      • Lapierre H.
      • Lobley G.E.
      • Doepel L.
      • Raggio G.
      • Rulquin H.
      • Lemosquet S.
      Triennial Lactation Symposium: Mammary metabolism of amino acids in dairy cows.
      ). Indeed, despite being lower on GR1+ILV compared with GR1+ALT, U:O of Arg was maintained >2 across all treatments, regardless of AA profile. Additionally, products of Lys catabolism formed in other tissues could have circulated to the mammary gland (
      • Bequette B.J.
      • Hanigan M.D.
      • Lapierre H.
      Mammary uptake and metabolism of amino acids by lactating ruminants.
      ;
      • Lapierre H.
      • Doepel L.
      • Milne E.
      • Lobley G.E.
      Responses in mammary and splanchnic metabolism to altered lysine supply in dairy cows.
      ). These, along with plasma peptides or breakdown products of mammary constituent protein, would not have been captured in our arteriovenous measurements but may have contributed to intramammary requirements for N and C skeletons (
      • Bequette B.J.
      • Backwell F.R.C.
      • MacRae J.C.
      • Lobley G.E.
      • Crompton L.A.
      • Metcalf J.A.
      • Sutton J.D.
      Effect of intravenous amino acid infusion on leucine oxidation across the mammary gland of the lactating goat.
      ).

      Mammary Lys Uptake may be Inhibited by High Concentrations of Ile and Leu

      Because milk protein yield did not differ between EAAC and GR1+ILV, lower mammary U:O of Lys with GR1+ILV infusion compared with EAAC arose from the lower mammary uptake of Lys. Arterial concentrations of Arg, Lys, and Thr were lower on GR1+ILV compared with EAAC, but only Lys uptake was affected. This suggests that Lys uptake may have been inhibited through a mechanism that did not affect other NB-group 2 AA. Arterial Lys concentration was not different between SAL, ILV, and GR1+ILV. Considering the similarly lower uptake of Lys observed on ILV infusion compared with EAAC, it is compelling to suggest that the presence of high levels of arterial BC-group 2 AA (i.e., 1,021 and 1,933 μM on GR1+ILV and ILV, respectively, compared with 753 μM on EAAC) inhibited Lys transport over the 5-d infusion period.
      • Yoder P.S.
      • Ruiz-Cortes T.
      • Castro J.J.
      • Hanigan M.D.
      Effects of varying extracellular amino acid profile on intracellular free amino acid concentrations and cell signaling in primary mammary epithelial cells.
      suggested that extracellular concentrations of AA in varying profiles affected Lys uptake into mammary cells measured in vitro. Cationic Na+-independent AA transport systems facilitate transfer of both Arg and Lys into bovine mammary cells (
      • Baumrucker C.R.
      Amino acid transport systems in bovine mammary tissue.
      ), and react with neutral AA, including Leu (
      • Shennan D.B.
      • Millar I.D.
      • Calvert D.T.
      Mammary-tissue amino acid transport systems.
      ). Lysine may also be transported into mammary cells using Na+-dependent systems ATBo,+ and y+LAT1, both identified in porcine mammary tissue (
      • Laspiur J.P.
      • Burton J.L.
      • Weber P.S.D.
      • Moore J.
      • Kirkwood R.N.
      • Trottier N.L.
      Dietary protein intake and stage of lactation differentially modulate amino acid transporter mRNA abundance in porcine mammary tissue.
      ;
      • Manjarin R.
      • Steibel J.R.
      • Zamora V.
      • Am-in N.
      • Kirkwood R.N.
      • Ernst C.W.
      • Weber P.S.
      • Taylor N.P.
      • Trottier N.L.
      Transcript abundance of amino acid transporters, β-casein and α-lactalbumin in mammary tissue of periparturient, lactating and postweaned sows.
      ), and Na+-independent LAT1 systems that have been identified in bovine mammary tissue (
      • Bionaz M.
      • Loor J.J.
      Gene networks driving bovine mammary protein synthesis during the lactation cycle.
      ), all of which are shared by Ile and Leu. When their arterial supply to mammary cells was high during ILV and GR1+ILV, it is possible that high intra- and extracellular concentrations of Ile and Leu reduced the transport of Lys into mammary cells via their shared transporters. The fact that Lys uptake may be more susceptible than Arg to competition for cellular transport further supports its apparent nonobligate intramammary metabolism compared with Arg (
      • Lapierre H.
      • Lobley G.E.
      • Doepel L.
      • Raggio G.
      • Rulquin H.
      • Lemosquet S.
      Triennial Lactation Symposium: Mammary metabolism of amino acids in dairy cows.
      ).

      Glucose Metabolism

      Whole-body glucose flux typically increases in response to increased absorptive supply of AA, but this has predominantly been investigated in response to complete AA profiles (
      • Clark J.H.
      • Spires H.R.
      • Derrig R.G.
      • Bennink M.R.
      Milk production, nitrogen utilization and glucose synthesis in lactating cows infused postruminally with sodium caseinate and glucose.
      ;
      • Lemosquet S.
      • Raggio G.
      • Lobley G.E.
      • Rulquin H.
      • Guinard-Flament J.
      • Lapierre H.
      Whole-body glucose metabolism and mammary energetic nutrient metabolism in lactating dairy cows receiving digestive infusions of casein and propionic acid.
      ;
      • Galindo C.E.
      • Ouellet D.R.
      • Pellerin D.
      • Lemosquet S.
      • Ortigues-Marty I.
      • Lapierre H.
      Effect of amino acid or casein supply on whole-body, splanchnic, and mammary glucose kinetics in lactating dairy cows.
      ). We hypothesized that differences in affinity of certain AA groups for hepatic metabolism may affect glucose appearance during 5-d infusion of incomplete EAA profiles. Therefore, a secondary objective of this experiment was to determine if the profile of supplemented EAA affects WB Ra of glucose. Whole-body glucose Ra increased 8, 12, and 16% over SAL with EAAC, GR1+ILV, and GR1+ALT, respectively (numerical increases only for EAAC and GR1+ILV), and did not differ appreciably between ILV and SAL. Hepatic gluconeogenesis drives the increment in glucose appearance with increased MP supply (
      • Galindo C.E.
      • Ouellet D.R.
      • Pellerin D.
      • Lemosquet S.
      • Ortigues-Marty I.
      • Lapierre H.
      Effect of amino acid or casein supply on whole-body, splanchnic, and mammary glucose kinetics in lactating dairy cows.
      ). Liver net uptake of group 1 AA, particularly Met, His, and Phe, is stimulated by increased protein intake (
      • Raggio G.
      • Pacheco D.
      • Berthiaume R.
      • Lobley G.E.
      • Pellerin D.
      • Allard G.
      • Dubreuil P.
      • Lapierre H.
      Effect of level of metabolizable protein on splanchnic flux of amino acids in lactating dairy cows.
      ;
      • Cantalapiedra-Hijar G.
      • Lemosquet S.
      • Rodriguez-Lopez J.M.
      • Messad F.
      • Ortigues-Marty I.
      Diets rich in starch increase the posthepatic availability of amino acids in dairy cows fed diets at low and normal protein levels.
      ;
      • Omphalius C.
      • Lemosquet S.
      • Ouellet D.R.
      • Bahloul L.
      • Lapierre H.
      Postruminal infusions of amino acids or glucose affect metabolisms of splanchnic, mammary, and other peripheral tissues and drive amino acid use in dairy cows.
      ). The relative increases in WB Ra of glucose over SAL with EAAC, GR1+ILV, and GR1+ALT reflect the inclusion level of group 1 AA in those treatments, where His, Met, and Phe could have contributed to glucose appearance via hepatic gluconeogenesis. Body protein turnover and hepatic AA catabolism stimulated by glucagon in response to EAA infusions (
      • Danfær A.
      • Tetens V.
      • Agergaard N.
      Review and an experimental study on the physiological and quantitative aspects of gluconeogenesis in lactating ruminants.
      ) also could have provided endogenous sources of AA-C for gluconeogenesis, particularly during ILV infusion where DMI decreased 1.3 kg/d compared with SAL (
      • Nichols K.
      • Bannink A.
      • Dijkstra J.
      Energy and nitrogen balance of dairy cattle as affected by provision of different essential amino acid profiles at the same metabolizable protein supply.
      ) and the arterial concentration of Ala decreased relative to SAL. Liver extraction of glucogenic branched-chain AA Ile and Val is negligible across a range of MP supplies (
      • Raggio G.
      • Pacheco D.
      • Berthiaume R.
      • Lobley G.E.
      • Pellerin D.
      • Allard G.
      • Dubreuil P.
      • Lapierre H.
      Effect of level of metabolizable protein on splanchnic flux of amino acids in lactating dairy cows.
      ;
      • Omphalius C.
      • Lemosquet S.
      • Ouellet D.R.
      • Bahloul L.
      • Lapierre H.
      Postruminal infusions of amino acids or glucose affect metabolisms of splanchnic, mammary, and other peripheral tissues and drive amino acid use in dairy cows.
      ). As such, the relative contribution of infused EAA to gluconeogenesis during ILV infusion was expected to have been less compared with the treatments supplying other EAA, as Ile and Val would first be exposed to utilization by extrahepatic tissues before their catabolic products would be used for gluconeogenesis in the liver (
      • Brosnan J.T.
      • Brosnan M.E.
      Branched-chain amino acids: Enzyme and substrate regulation.
      ). Catabolism of endogenous AA in support of glucose synthesis during ILV infusion agrees with the lowest N efficiency and plasma insulin concentration observed on this treatment (
      • Nichols K.
      • Bannink A.
      • Dijkstra J.
      Energy and nitrogen balance of dairy cattle as affected by provision of different essential amino acid profiles at the same metabolizable protein supply.
      ), as reduced plasma insulin favors proteolysis and gluconeogenesis.
      The majority of WB glucose flux in a lactating dairy cow is used by the mammary glands for milk synthesis (
      • Bickerstaffe R.
      • Annison E.F.
      • Linzell J.L.
      The metabolism of glucose, acetate, lipids and amino acids in lactating dairy cows.
      ). Mammary glucose uptake accounted for on average 76% of WB Ra of glucose across all treatments, which is consistent with
      • Galindo C.E.
      • Ouellet D.R.
      • Pellerin D.
      • Lemosquet S.
      • Ortigues-Marty I.
      • Lapierre H.
      Effect of amino acid or casein supply on whole-body, splanchnic, and mammary glucose kinetics in lactating dairy cows.
      who observed that 73% of WB Ra of glucose was used by the mammary gland during infusions of AA or casein. Lactose synthesis accounts for the majority of mammary-sequestered glucose, and oxidation of the remaining glucose facilitates energy and precursor supply for synthesis of TAG and proteins (
      • Bickerstaffe R.
      • Annison E.F.
      • Linzell J.L.
      The metabolism of glucose, acetate, lipids and amino acids in lactating dairy cows.
      ;
      • Xiao C.T.
      • Cant J.P.
      Relationship between glucose transport and metabolism in isolated bovine mammary epithelial cells.
      ). Mammary glucose uptake sufficiently covered theoretical glucose requirements for lactose and fat synthesis across all treatments in the current study, and mammary glucose balances did not differ from zero. In nonmammary tissues, postabsorptive glucose will be used predominantly for oxidation and TAG synthesis (
      • Danfær A.
      • Tetens V.
      • Agergaard N.
      Review and an experimental study on the physiological and quantitative aspects of gluconeogenesis in lactating ruminants.
      ). Whole-body glucose oxidation accounted for on average 17% of WB Ra of glucose, which agrees with the proportion of WB glucose flux directed to oxidation observed by
      • Bauman D.E.
      • Peel C.J.
      • Steinhour W.D.
      • Reynolds P.J.
      • Tyrrell H.F.
      • Brown A.C.G.
      • Haaland G.
      Effect of bovine somatotropin on metabolism of lactating dairy cows: Influence on rates of irreversible loss and oxidation of glucose and nonesterified fatty acids.
      ; 17.4%) in dairy cattle consuming a 15.9% CP diet, and slightly higher than that observed by
      • Clark J.H.
      • Spires H.R.
      • Derrig R.G.
      • Bennink M.R.
      Milk production, nitrogen utilization and glucose synthesis in lactating cows infused postruminally with sodium caseinate and glucose.
      during abomasal infusion of water (13.4%) or sodium caseinate (7.8%). Underlying our WB glucose oxidation estimate is the assumption that 30% of 13C was retained (
      • Junghans P.
      • Voigt J.
      • Jentsch W.
      • Metges C.C.
      • Derno M.
      The 13C bicarbonate dilution technique to determine energy expenditure in young bulls validated by indirect calorimetry.
      ) and did not appear in the 13CO2 measurements over the 24-h time period analyzed. Bicarbonate formation will vary with energy metabolism across physiological conditions; thus, WB glucose oxidation may be over- or underestimated depending on the dynamics of bicarbonate metabolism under the conditions of this experiment. Interestingly, negligible oxidation of mammary glucose uptake (i.e., glucose balance being numerically very close to zero) was observed with GR1+ILV infusion, and this treatment also produced numerically the lowest WB glucose oxidation as a proportion of WB Ra (15%). Because the mammary gland is the greatest net user of glucose in the body of a dairy cow, particularly in early to mid-lactation, it follows that intramammary glucose oxidation would influence total CO2 produced from glucose at the WB level.
      Bovine mammary cells are unable to convert gluconeogenic substrates to glucose due to the virtual absence of glucose-6-phosphatase in mammary tissue (
      • Scott R.A.
      • Bauman D.E.
      • Clark J.H.
      Cellular gluconeogenesis by lactating bovine mammary tissue.
      ). Thus, the glucose moiety of milk lactose primarily arises from free glucose taken up by the gland, but synthesis of the galactose moiety is possible in part from nonglucose hexose phosphate intermediates via the pentose phosphate pathway (
      • Wood H.G.
      • Peeters G.J.
      • Verbeke R.
      • Lauryssens M.
      • Jacobson B.
      Estimation of the pentose cycle in the perfused cow's udder.
      ). However, the contribution of nonglucose precursors to milk galactose synthesis is apparently not fixed, with ratios of galactose IE to glucose IE of 0.52 and 0.83 reported by
      • Maxin G.
      • Ouellet D.R.
      • Lapierre H.
      Contribution of amino acids to glucose and lactose synthesis in lactating dairy cows.
      and
      • Lapierre H.
      • Lemosquet S.
      • Ouellet D.R.
      Contribution of essential amino acids to glucose metabolism and lactose secretion in late lactation dairy cows.
      , respectively, in response to postruminal EAA infusions. Using bovine mammary explants incubated with [U-13C] glucose,
      • Bequette B.J.
      • Sunny N.E.
      • El-Kadi S.W.
      • Owens S.L.
      Application of stable isotopes and mass isotopomer distribution analysis to the study of intermediary metabolism of nutrients.
      reported that up to 86% of galactose in lactose was synthesized de novo from nonglucose carbon sources, and estimated that as much as 12% of galactose was derived from EAA catabolism. The observed 13C enrichment of lactose fractions in the current study suggests that approximately 28% of milk galactose arose from nonglucose precursors, with or without EAA supplementation and regardless of EAA profile. It appears that even when mammary glucose supply does not limit milk lactose or fat synthesis, AA carbon contributes to galactose synthesis, and that this contribution remains relatively constant when the EAA profile of supplemental MP is varied.

      CONCLUSIONS

      When Arg, Lys, and Thr, or Ile, Leu, and Val were absent from 5-d postruminal EAA infusions where the other 7 EAA equalized the MP supply, intramammary catabolism of the present group 2 AA compensated for lower mammary uptake of the absent EAA. During infusion of ILV and GR1+ILV, intramammary catabolism of branched-chain AA and Arg likely compensated for the contribution of Lys to oxidation and NEAA synthesis, suggesting that Lys catabolism is nonobligate for milk protein synthesis in mammary glands of dairy cattle. Overall, these findings illustrate flexibility in mammary net uptake and intramammary utilization of group 2 AA for oxidation and amino-N exchanges to support milk component synthesis when the EAA profile of MP is incomplete with respect to casein. Further, compared with a saline control, the increase in WB Ra of glucose with GR1+ALT and the numerical increases with EAAC and GR1+ILV reflect the inclusion level of group 1 AA in those infusions. Across all treatments, mammary gland glucose uptake accounted for on average 76% of WB Ra of glucose, and sufficiently covered mammary glucose requirements for milk component synthesis. Finally, on average 28% of milk galactose arose from nonglucose precursors, regardless of EAA profile infused.

      ACKNOWLEDGMENTS

      This research was conducted by Wageningen University and Research (Wageningen Livestock Research, Wageningen, the Netherlands), commissioned and funded by the Ministry of Agriculture, Nature and Food Quality (The Hague, the Netherlands) within the framework of Policy Support Research theme ‘Feed4Foodure' (BO-31.03-005-001; TKI-AF12039B), and by the Vereniging Diervoederonderzoek Nederland (Rijswijk, the Netherlands). The authors gratefully acknowledge technical assistance from Sven Alferink, Marcel Heetkamp, Tamme Zandstra, and the animal caretakers of the experimental facilities of “Carus” (Wageningen University and Research, Wageningen, the Netherlands), and from Abby-Ann Redman and Marlies Schuldink (students of Wageningen University, Wageningen, the Netherlands). Ultra-performance liquid chromatography was performed by Colette Mustière (INRA PEGASE, Saint-Gilles, France) with support from Ajinomoto Animal Nutrition Europe (Paris, France). Consultation on calculation of WB glucose metabolism was provided by Hélène Lapierre and Jocelyne Renaud (Agriculture and Agri-Food Canada, Sherbrooke, Canada). The authors have not stated any conflicts of interest.

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