Advertisement
Research| Volume 102, ISSUE 5, P4094-4104, May 2019

Short-term lactation and mammary metabolism responses in lactating goats to graded removal of methionine from an intravenously infused complete amino acid mixture

Open ArchivePublished:March 01, 2019DOI:https://doi.org/10.3168/jds.2018-15643
Short-term lactation and mammary metabolism responses in lactating goats to graded removal of methionine from an intravenously infused complete amino acid mixture
Previous ArticlePhosphorus content of muscle tissue and muscle function in dairy cows fed a phosphorus-deficient diet during the transition period
Next ArticleFactors associated with the content of mammary-synthesized fatty acids in milk fat: A meta-analysis

      ABSTRACT

      To investigate the possible pathways of Met deficiency to depress milk protein synthesis, 4 lactating goats fitted with jugular vein, mammary vein, and carotid artery catheters and transonic blood flow detectors on the external pudic artery were used in a 4 × 4 Latin square experiment. Goats were fasted for 24 h followed by a 9-h intravenous infusion of an AA mixture plus glucose. Milk yield was recorded and samples were taken in h 2 to 8 of the infusion period, and mammary biopsy was performed in the last hour. Treatments were graded removal of Met from the infused AA mixture to achieve Met content in the infusate of 100 (complete), 60, 30, or 0% of that in casein. Graded Met removal decreased yield of milk, milk protein, and lactose linearly and tended to decrease yield of milk fat linearly. Milk protein yield decreased to 82, 78, and 69% that of complete mixture infusion, respectively, when the 60, 30, and 0% Met infusate was infused. Circulating Met decreased linearly with graded Met removal. Arterial and venous Met decreased to 36 and 23% that of complete mixture infusion, respectively, when all Met was removed out of the mixture. Concomitant with the decreased circulating concentration was a similar increase in mammary Met affinity as reflected by the linearly increased mammary Met clearance rate. The increased affinity plus the linearly increased mammary blood flow totally offset the negative effect of decreased circulating Met concentration on mammary Met uptake. The overall result was similar mammary Met uptakes across treatments ranging from 285.9 to 339.5 μmol/h. Mammary uptakes of the other AA measured were generally not affected by treatments except for a linearly decreased Thr uptake and a trend of linearly increased Glu uptake. Consistent with the behavior of an AA mainly catabolized in the liver and mainly used for protein synthesis in peripheral tissues, mammary uptake to milk output ratios of Met measured in the present study ranged from 1.25 to 1.49 and was not affected by treatments. For the other AA measured, the ratio of Thr was linearly decreased and that of Glu was linearly increased by graded Met removal. Graded Met removal linearly elevated circulating urea N and glucose concentrations, indicating enhanced whole-body catabolism of AA and hepatic gluconeogenesis. Treatments had no significant effects on circulating insulin, growth hormone, and the other hormones and metabolites measured. Phosphorylation status of eIF4E binding protein 1 tended to decrease linearly and that of p70S6k was linearly decreased by graded Met removal, indicating depressed signal in the intracellular mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway. In conclusion, results of the present study indicated that the mTORC1 pathway and whole-body AA catabolism rather than mammary uptake appeared the drivers for changes in milk protein synthesis in response to varying Met supply.

      Key words

      INTRODUCTION

      The conversion of dietary N into product is quite inefficient in lactating ruminants relative to that in growing pigs. Farm surveys indicated that the efficiency for growing pigs was about 40% (
      • Nahm K.H.
      Efficient feed nutrient utilization to reduce pollutants in poultry and swine manure.
      Crit. Rev. Environ. Sci. Technol. 2002; 32: 1-16
      ) and that for lactating cows was about 25% (
      • Hristov A.N.
      • Price W.J.
      • Shafii B.
      A meta-analysis examining the relationship among dietary factors, dry matter intake, and milk and milk protein yield in dairy cows.
      J. Dairy Sci. 2004; 87 (15328233): 2184-2196
      ). Though one can argue that N digestibility is lower in ruminants than that in monogastric animals due to dietary roughage, this only explains part of the difference. Nitrogen digestibility in growing pigs is about 20 points higher than that in lactating cows (∼85 vs. 65%;
      • Baker D.H.
      Advances in amino acid nutrition and metabolism of swine and poultry.
      in: Kornegay E.T. Nutrient Management of Food Animals to Enhance and Protect the Environment. CRC Press, Boca Raton, FL1996: 41-52
      ;
      • Lapierre H.
      • Berthiaume R.
      • Raggio G.
      • Thivierge M.C.
      • Doepel L.
      • Pacheco D.
      • Dubreuil P.
      • Lobley G.E.
      The route of absorbed nitrogen into milk protein.
      Anim. Sci. 2005; 80: 11-22
      ), which explains about half of the difference in dietary N conversion efficiency. Postabsorptive N efficiency in lactating ruminants is also lower than that of growing pigs. Although the conversion efficiency of MP in growing pigs fed diets with well-balanced AA could reach 87% (
      • Baker D.H.
      Advances in amino acid nutrition and metabolism of swine and poultry.
      in: Kornegay E.T. Nutrient Management of Food Animals to Enhance and Protect the Environment. CRC Press, Boca Raton, FL1996: 41-52
      ), it appears to be about 40% in lactating cows (
      • Hanigan M.D.
      • Cant J.P.
      • Weakley D.C.
      • Beckett J.L.
      An evaluation of post absorptive protein and amino acid metabolism in the lactating dairy cow.
      J. Dairy Sci. 1998; 81 (9891282): 3385-3401
      ;
      • NRC
      Nutrient Requirements of Dairy Cattle.
      7th rev. ed. Natl. Acad. Press, Washington, DC2001
      ;
      • Lapierre H.
      • Lobley G.E.
      • Ouellet D.R.
      • Doepel L.
      • Pacheco D.
      Amino acid requirements for lactating dairy cows: Reconciling predictive models and biology.
      in: Proc. Cornell Nutrition Conference for Feed Manufacturers. Department of Animal Science, Cornell University, Ithaca, NY2007: 39-59
      ). Considering the limitations in increasing dietary N digestibility in lactating cows, improving postabsorptive N efficiency without sacrificing milk yield is therefore a major strategy to increase overall dietary N efficiency.
      The concept of efficiency implies inevitable losses of substrates during a process. With respect to the incorporation of AA into protein, the process per se results in no loss of AA. The increased loss of AA must occur elsewhere, such as the liver, portal-drained viscera, peripheral tissues, and metabolic pathways other than only milk protein synthesis in the mammary glands (
      • Berthiaume R.
      • Thivierge M.C.
      • Lobley G.E.
      • Dubreuil P.
      • Babkine M.
      • Lapierre H.
      Effect of a jugular infusion of essential amino acids on splanchnic metabolism in dairy cows fed a protein deficient diet.
      J. Dairy Sci. 2002; 85 (Abstr.): 72-73
      ;
      • Lapierre H.
      • Berthiaume R.
      • Raggio G.
      • Thivierge M.C.
      • Doepel L.
      • Pacheco D.
      • Dubreuil P.
      • Lobley G.E.
      The route of absorbed nitrogen into milk protein.
      Anim. Sci. 2005; 80: 11-22
      ). Decades of research efforts have enabled us to sketch out a general but far from detailed picture of postabsorptive AA metabolism in lactating ruminants (see review by
      • Lapierre H.
      • Berthiaume R.
      • Raggio G.
      • Thivierge M.C.
      • Doepel L.
      • Pacheco D.
      • Dubreuil P.
      • Lobley G.E.
      The route of absorbed nitrogen into milk protein.
      Anim. Sci. 2005; 80: 11-22
      ). Losses of total AA during absorption are proportional to MP supply (
      • Hanigan M.D.
      • Reynolds C.
      • Humphries D.
      • Lupoli B.
      • Sutton J.
      A model of net amino acid absorption and utilization by the portal-drained viscera of the lactating dairy cow.
      J. Dairy Sci. 2004; 87 (15545389): 4247-4268
      ;
      • Lapierre H.
      • Berthiaume R.
      • Raggio G.
      • Thivierge M.C.
      • Doepel L.
      • Pacheco D.
      • Dubreuil P.
      • Lobley G.E.
      The route of absorbed nitrogen into milk protein.
      Anim. Sci. 2005; 80: 11-22
      ) but there is variation in gut-associated losses among individual AA (
      • Berthiaume R.
      • Dubreuil P.
      • Stevenson M.
      • McBride B.W.
      • Lapierre H.
      Intestinal disappearance, mesenteric and portal appearance of amino acids in dairy cows fed ruminally protected methionine.
      J. Dairy Sci. 2001; 84 (11210033): 194-203
      ). Except for the branched-chain AA, the liver is responsible for removing excess AA to avoid toxicity while allowing passage of AA needed to support anabolism in peripheral tissues. Net portal absorption only contributes 5 to 20% of the total individual AA inflow into the liver of dairy cows with the remainder coming from arterial supply. With each pass of arterial blood, only a small proportion of AA is removed by the liver (1 to 9%), ensuring access to absorbed AA by the peripheral tissues. Amino acids not used by the peripheral tissues are recycled to the splanchnic bed, and the repeated cycling through the splanchnic tissues results in removal of the excess (
      • Lapierre H.
      • Berthiaume R.
      • Raggio G.
      • Thivierge M.C.
      • Doepel L.
      • Pacheco D.
      • Dubreuil P.
      • Lobley G.E.
      The route of absorbed nitrogen into milk protein.
      Anim. Sci. 2005; 80: 11-22
      ). This is demonstrated in the experiments of
      • Wray-Cahen D.
      • Metcalf J.
      • Backwell F.
      • Bequette B.
      • Brown D.
      • Sutton J.
      • Lobley G.
      Hepatic response to increased exogenous supply of plasma amino acids by infusion into the mesenteric vein of Holstein-Friesian cows in late gestation.
      Br. J. Nutr. 1997; 78 (9497443): 913-930
      ,
      • Berthiaume R.
      • Thivierge M.C.
      • Lobley G.E.
      • Dubreuil P.
      • Babkine M.
      • Lapierre H.
      Effect of a jugular infusion of essential amino acids on splanchnic metabolism in dairy cows fed a protein deficient diet.
      J. Dairy Sci. 2002; 85 (Abstr.): 72-73
      , and
      • Hanigan M.D.
      • Reynolds C.
      • Humphries D.
      • Lupoli B.
      • Sutton J.
      A model of net amino acid absorption and utilization by the portal-drained viscera of the lactating dairy cow.
      J. Dairy Sci. 2004; 87 (15545389): 4247-4268
      , where either intestinal, arterial, or jugular AA infusions resulted in a dramatic increase in hepatic removal of AA.
      Contrary to the splanchnic bed, the affinity of the mammary glands for EAA is relatively high (
      • Arriola Apelo S.I.
      • Knapp J.R.
      • Hanigan M.D.
      Invited review: Current representation and future trends of predicting amino acid utilization in the lactating dairy cow.
      J. Dairy Sci. 2014; 97 (24767883): 4000-4017
      ), resulting in 25 to 65% or more of AA removed in each pass, thus reducing recycling to the splanchnic tissues. It has been demonstrated that the clearance by mammary is variable and driven by mammary demand, primarily for milk protein synthesis (
      • Hanigan M.D.
      • France J.
      • Crompton L.A.
      • Bequette B.J.
      Evaluation of a representation of the limiting amino acid theory for milk protein synthesis.
      in: McNamara J.P. France J. Beever D.E. Modelling Nutrient Utilization in Farm Animals. CABI, Wallingford, UK2000: 127-144
      ;
      • Mackle T.R.
      • Dwyer D.A.
      • Ingvartsen K.L.
      • Chouinard P.Y.
      • Ross D.A.
      • Bauman D.E.
      Effects of insulin and postruminal supply of protein on use of amino acids by the mammary gland for milk protein synthesis.
      J. Dairy Sci. 2000; 83 (10659969): 93-105
      ;
      • Bequette B.J.
      • Kyle C.
      • Crompton L.
      • Buchan V.
      • Hanigan M.
      Insulin regulates milk production and mammary gland and hind-leg amino acid fluxes and blood flow in lactating goats.
      J. Dairy Sci. 2001; 84 (11210039): 241-255
      ). Thus, increased demand within the mammary can stimulate affinity and uptake, but increased external supply is at least partially resisted through decreased mammary affinity (
      • Ying F.
      • Lin X.Y.
      • Ma W.M.
      • Chi H.L.
      • Yan Z.G.
      • Song Y.F.
      • Wang Z.H.
      Metabolic responses to the deficiency of Lys, Arg, Met, or His in the mammary gland of lactating goats.
      Small Rumin. Res. 2013; 113: 219-230
      ) and uptake can be mostly maintained in the face of decreased supply through increased affinity (
      • Guo C.L.
      • Li Y.T.
      • Lin X.Y.
      • Hanigan M.D.
      • Yan Z.G.
      • Hu Z.Y.
      • Hou Q.L.
      • Jiang F.G.
      • Wang Z.H.
      Effects of graded removal of lysine from an intravenously infused amino acid mixture on lactation performance and mammary amino acid metabolism in lactating goats.
      J. Dairy Sci. 2017; 100 (28434735): 4552-4564
      ). These concepts are all captured in the model of
      • Hanigan M.D.
      • France J.
      • Crompton L.A.
      • Bequette B.J.
      Evaluation of a representation of the limiting amino acid theory for milk protein synthesis.
      in: McNamara J.P. France J. Beever D.E. Modelling Nutrient Utilization in Farm Animals. CABI, Wallingford, UK2000: 127-144
      , which provided greater prediction accuracy and precision than a model with static uptake kinetics.
      Based on these concepts, it should be possible to decrease postabsorptive AA losses through nutritional regulation by stimulating use in mammary for milk protein synthesis while reducing the excess supply of each AA. Mechanistic target of rapamycin complex 1 (mTORC1) is at the center of intracellular signaling pathway to regulate protein synthesis (
      • Kim S.G.
      • Buel G.R.
      • Blenis J.
      Nutrient regulation of the mTOR complex 1 signaling pathway.
      Mol. Cells. 2013; 35 (23694989): 463-473
      ). Its activity in mammary epithelial cells is stimulated by increased glucose and acetate supply through the AMPK pathway (
      • Appuhamy J.A.D.R.N.
      Regulatory roles of essential amino acids, energy, and insulin in mammary cell protein synthesis.
      Animal Sciences, Virginia Polytechnic Institute and State University, Blacksburg2010
      ), by elevated circulating insulin through the AKT pathway (
      • Appuhamy J.A.
      • Knapp J.R.
      • Becvar O.
      • Escobar J.
      • Hanigan M.D.
      Effects of jugular-infused lysine, methionine, and branched-chain amino acids on milk protein synthesis in high-producing dairy cows.
      J. Dairy Sci. 2011; 94 (21426986): 1952-1960
      ), and by the direct actions of several EAA including Leu, Ile, Met, and Thr (
      • Arriola Apelo S.I.
      • Knapp J.R.
      • Hanigan M.D.
      Invited review: Current representation and future trends of predicting amino acid utilization in the lactating dairy cow.
      J. Dairy Sci. 2014; 97 (24767883): 4000-4017
      ;
      • Liu G.M.
      • Hanigan M.D.
      • Lin X.Y.
      • Zhao K.
      • Jiang F.G.
      • White R.R.
      • Wang Y.
      • Hu Z.Y.
      • Wang Z.H.
      Methionine, leucine, isoleucine, or threonine effects on mammary cell signaling and pup growth in lactating mice.
      J. Dairy Sci. 2017; 100 (28237591): 4038-4050
      ), respectively. Some individual AA, such as Phe (
      • Güttler F.
      • Kühl C.
      • Pedersen L.
      • Påby P.
      Effects of oral phenylalanine load on plasma glucagon, insulin, AA and glucose concentrations in man.
      Scand. J. Clin. Lab. Invest. 1978; 38 (663549): 255-260
      ) and Met (
      • Stone K.P.
      • Wanders D.
      • Orgeron M.
      • Cortez C.C.
      • Gettys T.W.
      Mechanisms of increased in vivo insulin sensitivity by dietary methionine restriction in mice.
      Diabetes. 2014; 63 (24947368): 3721-3733
      ), have been reported to be insulinotropic. Methionine is primarily catabolized in the liver, and its net splanchnic outflow is similar to that of milk protein output (
      • Lapierre H.
      • Berthiaume R.
      • Raggio G.
      • Thivierge M.C.
      • Doepel L.
      • Pacheco D.
      • Dubreuil P.
      • Lobley G.E.
      The route of absorbed nitrogen into milk protein.
      Anim. Sci. 2005; 80: 11-22
      ). Thus, it may be possible to elevate postabsorptive efficiency of AA for milk protein synthesis by modulating their circulating profile.
      The objective of this work was to evaluate the relative importance of the various pathways associated with Met catabolism and use for milk protein synthesis when animals were subjected to varying Met supply whereas that of the other AA was held constant in a 9-h infusion experiment. The hypothesis was that mTORC1 pathway and catabolism of AA contributed to short-term changes in milk protein synthesis in response to varying Met supply.

      MATERIALS AND METHODS

      Animals and Diet

      Four multiparous Laoshan dairy goats averaging (mean ± SD) 108 ± 12 DIM and 38 ± 6 kg of BW were used. All animal handling and surgical procedures were approved by the Institutional Animal Care and Use Committee of Shandong Agricultural University. The right carotid artery was elevated to a subcutaneous position, and a blood flow detector was surgically fitted to the right external pudic artery as previously described (
      • Guo C.L.
      • Li Y.T.
      • Lin X.Y.
      • Hanigan M.D.
      • Yan Z.G.
      • Hu Z.Y.
      • Hou Q.L.
      • Jiang F.G.
      • Wang Z.H.
      Effects of graded removal of lysine from an intravenously infused amino acid mixture on lactation performance and mammary amino acid metabolism in lactating goats.
      J. Dairy Sci. 2017; 100 (28434735): 4552-4564
      ). Goats were housed in individual stalls before and after each experimental period, and in individual metabolism stalls during each period. The goats were fed and milked twice daily at 0800 and 1800 h except as noted below during the infusion.
      The pelleted diet that was fed was predicted to provide 10.86 MJ of ME and 49.44 g of MP/kg of DM according to
      • AFRC
      Energy and Protein Requirements of Ruminants.
      CAB International, Wallingford, UK1993
      , Table 1). The daily amount of the diet offered was adjusted to maintain a 5% refusal rate. Goats were allowed free access to water throughout the experiment.
      Table 1Ingredients, nutrients, and EAA compositions of the pelleted diet
      ItemValue
      Ingredient, % of DM
       Corn14.42
       Soybean meal13.61
       Wheat bran4.86
       Alfalfa hay33.78
       Leymus chinensis28.85
       Calcium carbonate0.19
       Calcium bicarbonate3.73
       Salt0.38
       Premix
      Premix contained (per kg of DM): 100 kIU of vitamin A, 250 kIU of vitamin D3, 2,400 mg of vitamin E, 2,000 mg of nicotinic acid, 2,000 mg of Fe, 3,000 mg of Mn, 3,000 mg of Cu, 14,000 mg of Zn, 100 mg of Se, 180 mg of I, and 40 mg of Co.
      		0.18
      Nutrient
       ME,
      Data were calculated according to NRC models (NRC, 2001) based on contents of EAA and RUP of the ingredients measured in our laboratory.
      MJ/kg of DM      		10.86
       MP,
      Data were calculated according to NRC models (NRC, 2001) based on contents of EAA and RUP of the ingredients measured in our laboratory.
      g/kg of DM      		103
       CP,
      Laboratory-determined data.
      % of DM      		16.41
       NDF,
      Laboratory-determined data.
      % of DM      		30.84
       ADF,
      Laboratory-determined data.
      % of DM      		18.89
      EAA,
      Calculated data from a laboratory-determined data set of AA composition of feed ingredients.
      g/kg of DM unless noted otherwise
       Lys10.31
       Met2.69
       His4.32
       Arg10.88
       Thr7.02
       Val9.04
       Ile10.85
       Leu11.48
       Phe9.59
       Trp0.61
       Lys, % of MP10.0
       Met, % of MP2.6
      1 Premix contained (per kg of DM): 100 kIU of vitamin A, 250 kIU of vitamin D3, 2,400 mg of vitamin E, 2,000 mg of nicotinic acid, 2,000 mg of Fe, 3,000 mg of Mn, 3,000 mg of Cu, 14,000 mg of Zn, 100 mg of Se, 180 mg of I, and 40 mg of Co.
      2 Data were calculated according to NRC models (
      • NRC
      Nutrient Requirements of Dairy Cattle.
      7th rev. ed. Natl. Acad. Press, Washington, DC2001
      ) based on contents of EAA and RUP of the ingredients measured in our laboratory.
      3 Laboratory-determined data.
      4 Calculated data from a laboratory-determined data set of AA composition of feed ingredients.

      Experimental Design and Procedure

      The experimental design was a 4 × 4 Latin square with four 15-d periods. The complete AA mixture that was used was formulated according to the profile of casein (Table 2). Treatments were graded removal of Met from the complete mixture. This was achieved by substitution of Met with glutamate on a molar basis. The final Met content in the 4 infusates were 100 (complete), 60, 30, and 0% of that of in the complete mixture. Infusates were prepared and infusion was performed according to the procedures described previously (
      • Guo C.L.
      • Li Y.T.
      • Lin X.Y.
      • Hanigan M.D.
      • Yan Z.G.
      • Hu Z.Y.
      • Hou Q.L.
      • Jiang F.G.
      • Wang Z.H.
      Effects of graded removal of lysine from an intravenously infused amino acid mixture on lactation performance and mammary amino acid metabolism in lactating goats.
      J. Dairy Sci. 2017; 100 (28434735): 4552-4564
      ).
      Table 2AA profile of the complete AA mixture
      Adapted from Bequette et al. (1996).
      Item, g/100 g of total AAValue
      EAA
       Lys7.49
       Met2.82
       Leu8.89
       Ile4.61
       Val6.24
       Thr3.86
       Phe4.75
       His2.82
       Arg3.57
       Trp1.49
      NEAA
       Ala2.82
       Gly1.78
       Glu9.95
       Gln9.80
       Asn3.40
       Asp6.83
       Ser5.05
       Pro8.32
       Cys0.59
       Tyr4.90
      1 Adapted from
      • 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.
      J. Dairy Sci. 1996; 79 (9029360): 2217-2224
      .
      Two days before each experimental period, indwelling catheters were placed in the left jugular vein, right carotid artery, and right mammary vein of each goat. Goats were fasted for 35 h with infusions of AA and glucose during the final 9 h. Amounts of total AA infused during the 9 h, which ranged from 37 to 58 g, were calculated to match the MP requirement of each goat according to
      • AFRC
      Energy and Protein Requirements of Ruminants.
      CAB International, Wallingford, UK1993
      based on their respective milk protein yield recorded for the successive 3 d before the infusion. The amount of glucose infused was calculated to provide 3.6 mg of glucose/kg of BW per min according
      • Guo C.L.
      • Li Y.T.
      • Lin X.Y.
      • Hanigan M.D.
      • Yan Z.G.
      • Hu Z.Y.
      • Hou Q.L.
      • Jiang F.G.
      • Wang Z.H.
      Effects of graded removal of lysine from an intravenously infused amino acid mixture on lactation performance and mammary amino acid metabolism in lactating goats.
      J. Dairy Sci. 2017; 100 (28434735): 4552-4564
      . Feeding was restored after completion of each infusion. Goats were moved into individual stalls and rested for 10 d between each experimental period.

      Sampling and Analysis

      Goats were milked at 1 and 8 h after the start of the infusion. Milk collected at the second milking (milk secreted during the 7-h interval) was collected, weighed, recorded, and sampled for later analysis. Before each milking, 1 IU of oxytocin was intravenously injected into each goat to allow for complete milk removal (
      • 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.
      J. Dairy Sci. 1996; 79 (9029360): 2217-2224
      ). Milk samples were divided into 2 subsamples: one for AA determination after hydrolysis using an AA analyzer (Type 835–50, Hitachi, Tokyo, Japan), and the other for milk composition analysis using an infrared milk composition analyzer (Type 78110, Foss, Hillerød, Denmark). Milk samples were stored at −20°C with hydrogen peroxide for later analysis. The samples for AA analysis were sent to be hydrolyzed with hydrochloric acid at a final concentration of 4 M. The hydrolysate was filtrated through 0.2-μm film before being loaded into the AA analyzer.
      Five milliliters of blood was simultaneously collected from the carotid artery and mammary vein into heparinized evacuated tubes at hourly intervals from 1 to 8 h after the start of the infusion. Animals were kept in a standing position during blood sampling. The 7 samples taken from each blood vessel were pooled and stored at −20°C for later analysis of free AA concentrations using an AA analyzer (Type 835–50, Hitachi). Blood sample was deproteinized by mixing with equal amounts of 10% sulfosalicylic acid, kept at 4°C overnight, and centrifuged at 15,000 × g for 20 min at 4°C. The supernatant withdrawn was passed through a 0.2-μm film before being loaded into the AA analyzer. Plasma was prepared from an aliquot of each blood sample taken from the mammary vein and used for later analysis of metabolites and hormones. Plasma urea N and glucose were determined by an automatic biochemical analyzer (Type 7020, Hitachi), total protein and nitric oxide (NO) were determined by commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), and IGF-I, insulin, glucagon, and growth hormone (GH) were determined by RIA using commercial assay kits (Tianjin Jiuding Biochemical Engineering Co. Ltd., Tianjin, China). Mammary blood flow during each infusion period was measured using a transit time ultrasonic flow system (Type MC6PSS-LS-WCS10-GC, Transonic Systems Inc., Ithaca, NY) calibrated according to the manufacturer's instructions.
      Biopsies were taken from the upper portion of the left mammary gland following the second milking (between 8 and 9 h of the infusion) during each period. Mammary biopsies were performed using a 12-gauge, 16-cm MG1522 Bard Magnum reusable core biopsy instrument (Bard Medical Division, 8195 Industrial Boulevard, Covington, GA). About 0.1 g of mammary tissue was sampled after subcutaneous injection of lidocaine hydrochloride and skin incision at the sampling position. Mammary tissue was immediately rinsed in ice-cold saline, frozen, and stored in liquid N for later analysis of phosphorylation of p70S6k, eIF4E binding protein 1 (4EBP1), and eIF2α by Western blotting.

      Calculation and Statistics

      Mammary clearance rates (KClearance;
      • 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.
      Br. J. Nutr. 1998; 79 (9505804): 63-78
      ) were calculated to assess tissue affinity for blood metabolites:
      KClearance, L/time = [(CA − CV)/CV] × BFMam,


      where CA = arterial concentration of the measured AA, CV = venous concentration of the measured AA, and BFMam = mammary blood flow. Mammary uptake (UMam; μmol/h) of individual AA was calculated as
      UMam = (CA – CV) × BFMam × 2.


      The flux was multiplied by 2 to express on a whole-tissue basis. Mammary uptake to milk output ratios (U:O) for individual AA were calculated as mammary uptake of the AA divided by the amount secreted in milk during the 7 h between milkings.
      Data were subjected to mixed-effect regression analysis by SAS 9.0 (SAS Institute Inc., Cary, NC). The statistical model used was
      Yijk = μ + αi + βj + γk + eijk,


      where Yijk = the observed value, μ = the overall mean, αi = the class effect of ith experimental period, βj = the continuous effect of Met dose, γk = the random effect of the kth animal, and eijk = the random error. Both linear and quadratic effects of Met dose were tested initially. When quadratic effects were nonsignificant, they were removed from the model. Significance was declared at P ≤ 0.05, and a tendency was declared at P ≤ 0.1.

      RESULTS

      Milk Yields and Composition

      Removal of Met from the infused mixture decreased the yields of milk, milk protein, and lactose linearly (P < 0.01, Table 3). A tendency was observed for milk fat yield and milk protein content to decrease linearly with graded removal of Met (P < 0.1). Milk fat to protein ratio was linearly increased by graded Met removal (P < 0.05).
      Table 3Effects of graded removal of Met from the AA mixture infused into jugular vein on lactation performance of lactating goats
      ItemMet concentration in the infusateSEP-value
      100%60%30%0%LinearQuadratic
      Milk yield, g/7 h405.0364.5340.3321.079.40.00030.568
      Milk protein, %4.414.174.283.960.380.0800.782
       g/7 h17.3614.2913.5811.991.85<0.00010.325
      Milk fat, %5.165.255.635.950.700.1470.688
       g/7 h20.6019.0118.5717.293.550.0620.979
      Lactose, %4.464.664.784.660.210.1530.203
       g/7 h18.4717.4216.5215.334.500.0090.767
      Fat/protein1.191.301.371.510.180.0300.782
      MUN38.7234.5438.8636.812.410.7940.498

      Arterial Free AA

      Methionine removal decreased arterial Met concentration linearly (P < 0.01, Table 4). Removing all Met from the infusate decreased arterial Met to about one-third that of full mixture infusion. Arterial concentrations of the other measured AA were generally unaffected by treatments except for that of His, Ser, and Gly. Arterial His tended to increase linearly; Gly tended to increase quadratically (P < 0.1); and Ser declined linearly and quadratically (P < 0.05).
      Table 4Effects of graded removal of Met from the AA mixture infused into the jugular vein on AA concentrations in the plasma of carotid artery of lactating goats
      Item, μMMet concentration in the infusateSEP-value
      100%60%30%0%LinearQuadratic
      EAA
       Met35.6618.9113.4312.744.300.0030.120
       Lys320.0254.7280.0266.737.20.1710.251
       Thr188.5158.4159.2196.723.70.8760.117
       Val240.7244.3259.7243.821.00.7330.645
       Ile83.1989.7489.9382.737.790.9750.272
       Leu131.1141.3143.8133.115.80.8310.421
       Phe85.4791.4094.8378.218.860.6620.152
       His68.9479.6686.1084.276.810.0900.431
       Arg168.4159.4160.0161.814.290.7260.708
      NEAA
       Ser84.1966.2475.7685.319.540.0320.031
       Glu355.6310.9342.4341.237.280.8420.345
       Gly594.9650.8656.9585.356.170.9800.072
       Ala181.3177.2196.6196.016.00.3630.820
       Cys60.3767.2165.6870.415.720.2400.875
       Tyr87.7585.5993.4189.247.090.6950.958
       Pro298.5316.8354.4324.226.070.3150.442

      Venous Free AA

      Graded Met removal decreased venous Met linearly (P < 0.01, Table 5), and a tendency for a quadratic effect (P < 0.1) was observed. Removing all Met from the infusate decreased venous Met to less than one-quarter that of full mixture infusion. Out of the other AA measured, graded Met removal increased venous His and Ala linearly, increased venous Ser linearly and quadratically (P < 0.05), tended to increase venous Thr and Cys linearly, tended to increase venous Phe quadratically, and tended to decrease venous Lys quadratically (P ≤ 0.10). Treatments had no significant effects on venous concentrations of the other AA measured.
      Table 5Effects of graded removal of Met from the AA mixture infused into jugular vein on AA concentrations in the plasma of mammary vein in lactating goats, μM
      ItemMet Concentration in the infusateSEP-value
      100%60%30%0%LinearQuadratic
      EAA
       Met26.8011.436.956.213.020.00080.051
       Lys271.4210.3241.7241.434.40.4320.100
       Thr140.1126.6146.5192.422.30.0950.102
       Val192.3203.8239.7212.820.70.2330.399
       Ile60.6757.0669.5564.436.770.4310.967
       Leu95.2389.95113.7104.514.00.3910.997
       Phe66.4777.4385.8870.888.270.4190.055
       His59.6065.7079.3978.107.730.0310.743
       Arg127.4125.1142.5142.99.210.1410.745
      NEAA
       Ser70.0060.1267.9879.1011.920.0240.040
       Glu309.8257.6294.7291.939.70.8180.342
       Gly488.7538.5597.5549.254.20.1410.236
       Ala126.6150.8166.5181.316.20.0160.893
       Cys49.9659.6360.5067.136.410.0680.877
       Tyr80.3577.7182.0785.577.400.5460.631
       Pro253.4270.8334.5299.336.00.1360.503

      Venous Metabolites, Hormones, and Mammary Blood Flow

      Graded Met removal increased venous urea N and glucose and BFMam linearly (P < 0.05, Table 6). Treatments tended to affect venous GH and NO quadratically in that venous GH varied in a down-up and NO varied in an up-down manner, respectively, with graded Met removal (P < 0.1).
      Table 6Effects of graded removal of Met from the AA mixture infused into jugular vein on mammary blood flow (BFMam) and concentrations of metabolites and hormones in the plasma of mammary vein in lactating goats
      Item
      TP = total protein; PRL = prolactin; GH = growth hormone.
      		Met concentration in the infusateSEP-value
      100%60%30%0%LinearQuadratic
      Urea N, mmol/L4.204.364.464.490.360.0300.593
      Glucose, mmol/L4.225.035.405.350.370.0080.136
      TP, mg/mL75.2772.5470.9770.503.600.1740.874
      IGF-1, ng/mL95.3186.8080.4184.7013.980.3330.554
      PRL2.572.281.771.680.630.1760.947
      GH, ng/mL0.290.140.100.240.090.5440.098
      Insulin, μIU/mL58.3262.8085.6661.4315.640.5280.317
      Glucagon, pg/mL155.5173.3139.1179.431.550.5650.546
      NO, μmol/L8.7513.4917.179.954.010.6440.095
      BFMam, L/7 h131.4142.1156.0174.732.540.00070.332
      1 TP = total protein; PRL = prolactin; GH = growth hormone.

      Mammary AA Clearance Rates

      Graded Met removal increased mammary clearance rates of circulating Met linearly (P < 0.01, Table 7). Relative to that of the full mixture infusion, the 30 and 0% Met infusions increased mammary Met clearance rates by approximately 5-fold. Mammary clearance rates of the other AA were not statistically affected by treatments except for a trend for decreased Gly removal (P < 0.1).
      Table 7Effects of graded removal of Met from the AA mixture infused into a jugular vein on mammary AA clearance rates (L/h) in lactating goats
      ItemMet concentration in the infusateSEP-value
      100%60%30%0%LinearQuadratic
      EAA
       Met5.8614.7228.6427.105.620.0020.389
       Lys2.784.233.252.980.950.9940.342
       Thr5.036.632.251.022.030.1080.374
       Val4.223.571.874.051.140.6450.303
       Ile6.3510.227.148.322.290.6070.343
       Leu6.7310.296.528.372.400.8220.569
       Phe5.253.142.633.231.660.2940.400
       His2.333.512.022.701.080.9700.715
       Arg4.814.823.124.421.310.4720.598
      NEAA
       Ser3.222.532.852.891.520.8810.771
       Glu2.293.553.834.721.220.1510.939
       Gly4.463.721.841.711.240.0810.943
       Ala9.212.613.782.962.980.1320.292
       Cys3.131.901.831.781.1350.3720.629
       Tyr1.321.423.741.171.120.6890.321
       Pro3.362.280.122.803.180.7470.583

      Mammary Uptakes of Circulating AA

      Graded Met removal did not change mammary uptake of circulating Met even though all the Met was removed out of the infusate (P > 0.1, Table 8). Graded Met removal decreased mammary uptake of circulating Thr linearly (P < 0.05) and tended to increase the uptake of circulating Glu linearly (P < 0.1). Mammary uptakes of the other AA measured were not statistically affected by treatments.
      Table 8Effects of graded removal of Met from the AA mixture infused into jugular vein on mammary AA uptakes in lactating goats, μmol/h
      ItemMet concentration in the infusateSEP-value
      100%60%30%0%LinearQuadratic
      EAA
       Met339.5285.9301.2336.9102.40.9820.553
       Lys1,5001,5551,6841,4825480.9530.777
       Thr1,4951,1816792754190.0340.778
       Val1,5671,5289171,6544420.8160.402
       Ile8281,1981,0071,0113390.4610.138
       Leu1,2881,8631,5391,6015630.4710.231
       Phe729.7416.2446.1432.8222.30.2780.453
       His278.0429.7303.4343.2126.30.8140.525
       Arg605.4564.3445.3547.3173.90.5840.644
      NEAA
       Ser416.5153.5313.3375.0175.70.9690.329
       Glu1,2351,7472,1662,6406430.0580.922
       Gly2,11020501,0369316460.1050.830
       Ala2,5977101,0098598850.1470.280
       Cys317.0223.2220.9168.31260.3900.894
       Tyr198.4209.6517.1202.7142.50.5940.345
       Pro1,562566−9931,6982,1570.8650.422

      Mammary Uptake to Output Ratio of AA

      Mammary uptake to milk protein output ratios for Met ranged from 1.24 to 1.49 and were not affected by treatment (P > 0.1, Table 9). The U:O of Thr was linearly decreased and that of Glu was linearly increased by graded removal of Met from the infused AA mixture (P ≤ 0.05). The U:O of Thr decreased to 0.32 in the treatment with all Met removed, which implied the amount of Thr extracted in the form of free AA into mammary glands was less the amount secreted into milk protein and the imbalance might be made up by other forms of Thr extracted, such as peptide-bound Thr. Treatments had no significant effects on the U:O of the other measured AA.
      Table 9Effects of graded removal of Met from the AA mixture infused into jugular vein on mammary uptake to output ratio (U:O) of AA in lactating goats
      ItemMet concentration in the infusateSEP-value
      100%60%30%0%LinearQuadratic
      EAA
       Met1.331.251.331.490.400.6910.668
       Lys2.602.893.702.841.080.7230.639
       Thr3.713.311.430.321.240.0420.630
       Val1.511.430.911.700.350.9890.304
       Ile2.443.883.223.500.730.2640.283
       Leu1.802.832.312.660.610.2650.434
       Phe1.961.171.151.150.510.2490.466
       His1.552.572.151.980.760.7380.428
       Arg3.924.363.154.280.980.9740.776
      NEAA
       Ser1.040.280.750.970.420.9750.236
       Glu0.771.321.842.280.440.0160.951
       Gly10.3110.507.314.932.970.1550.570
       Ala6.402.383.102.602.060.1800.361
       Cys1.110.881.390.560.650.6910.675
       Tyr0.750.691.710.760.560.6230.473
       Pro2.121.361.042.002.190.9140.682

      Phosphorylation of Downstream Proteins in mTORC1 Pathway

      Graded removal of Met from the intravenously infused AA mixture tended to decrease phosphorylation status of 4EBP1 (P = 0.053) and decreased that of p70S6K linearly (P < 0.05). Treatments had no significant effect on the phosphorylation of eIF2.

      DISCUSSION

      Milk Yield and Composition

      Methionine and Lys are the 2 EAA that are frequently identified as limiting in the diets of lactating ruminants. It has been observed that increased postruminal supply of Met alone (
      • Guinard J.
      • Rulquin H.
      Effect of graded amount of duodenal infusions of methionine on the mammary uptake of major milk precursors in dairy cows.
      J. Dairy Sci. 1995; 78 (8598404): 2196-2207
      ;
      • Noftsger S.
      • St-Pierre N.R.
      Supplementation of methionine and selection of highly digestible rumen undegradable protein to improve nitrogen efficiency for milk production.
      J. Dairy Sci. 2003; 86: 958-969
      10.3168/jds.S0022-0302(03)73679-0
      ;
      • Chen Z.H.
      • Broderick G.A.
      • Luchini N.D.
      • Sloan B.K.
      • Devillard E.
      Effect of feeding different sources of rumen-protected methionine on milk production and N-utilization in lactating dairy cows.
      J. Dairy Sci. 2011; 94: 1978-1988
      10.3168/jds.2010-3578
      ;
      • Zhou Z.
      • Vailati-Riboni M.
      • Trevisi E.
      • Drackley J.K.
      • Luchini D.N.
      • Loor J.J.
      Better postpartal performance in dairy cows supplemented with rumen-protected methionine compared with choline during the peripartal period.
      J. Dairy Sci. 2016; 99: 1-17
      10.3168/jds.S0022-0302(83)82054-2
      ) or in combination with Lys (
      • Rogers J.A.
      • Krishnamoorthy U.
      • Sniffen C.J.
      Plasma amino acids and milk protein production by cows fed rumen-protected methionine and lysine.
      J. Dairy Sci. 1987; 70: 789-798
      10.3168/jds.S0022-0302(87)80075-9
      ;
      • Donkin S.S.
      • Varga G.A.
      • Sweeney T.F.
      • Muller L.D.
      Rumen-protected methionine and lysine: Effects on animal performance, milk protein yield, and physiological measures.
      J. Dairy Sci. 1989; 72: 1484-1491
      10.3168/jds.S0022-0302(89)79258-4
      ;
      • Misciattelli L.
      • Kristensen V.F.
      • Vestergaard M.
      • Weisbjerg M.R.
      • Sejrsen K.
      • Hvelplund T.
      Milk production, nutrient utilization, and endocrine responses to increased postruminal lysine and methionine supply in dairy cows.
      J. Dairy Sci. 2003; 86: 275-286
      10.3168/jds.S0022-0302(03)73606-6
      ;
      • Appuhamy J.A.
      • Knapp J.R.
      • Becvar O.
      • Escobar J.
      • Hanigan M.D.
      Effects of jugular-infused lysine, methionine, and branched-chain amino acids on milk protein synthesis in high-producing dairy cows.
      J. Dairy Sci. 2011; 94 (21426986): 1952-1960
      ;
      • Lee C.
      • Hristov A.N.
      • Cassidy T.W.
      • Heyler K.S.
      • Lapierre H.
      • Varga G.A.
      • de Veth M.J.
      • Patton R.A.
      • Parys C.
      Rumen-protected lysine, methionine, and histidine increase milk protein yield in dairy cows fed a metabolizable protein-deficient diet.
      J. Dairy Sci. 2012; 95: 6042-6056
      10.3168/jds.2012-5581
      ) promoted milk protein production.
      • NRC
      Nutrient Requirements of Dairy Cattle.
      7th rev. ed. Natl. Acad. Press, Washington, DC2001
      recommended that the optimal level of Met for lactating cows was 2.40% of dietary MP, which was supported by the results of
      • Cho J.
      • Overton T.R.
      • Schwab C.G.
      • Tauer L.W.
      Determining the amount of rumen-protected methionine supplement that corresponds to the optimal levels of methionine in metabolizable protein for maximizing milk protein production and profit on dairy farms.
      J. Dairy Sci. 2007; 90: 4908-4916
      10.3168/jds.2007-0314
      . However, decreased postruminal Met supply and decreased milk protein yield does not necessarily equate to decreased mammary uptake (
      • Ying F.
      • Lin X.Y.
      • Ma W.M.
      • Chi H.L.
      • Yan Z.G.
      • Song Y.F.
      • Wang Z.H.
      Metabolic responses to the deficiency of Lys, Arg, Met, or His in the mammary gland of lactating goats.
      Small Rumin. Res. 2013; 113: 219-230
      ). Therefore, Met supply may exert a portion of its effects on milk protein synthesis through circulating hormone concentrations and intracellular signaling in mammary epithelial cells rather than through mass action. Though Met deficiency decreased the yields of milk and milk protein (P < 0.001, Table 3), mammary uptakes of Met, total AA, and all the measured AA except for Thr and Glu were not affected in the present study (Table 7). The decreases in milk protein yield induced by Met deficiency, at least for the short term, could therefore hardly be attributed to an effect of mass action. Whether mass action plays an important part in decreasing milk protein yield under the scenario of long-term Met deficiency is a topic worth further investigation. More profound variations in systemic metabolism pathways, including those in the rumen, may occur under this situation.
      Supplementation of Met has been reported to promote milk fat production, and the study has been carried out to investigate the effects of Met on milk fat rather than milk protein productions (
      • Lundquist R.G.
      • Otterby D.E.
      • Linn J.G.
      Influence of three concentrations of DL-methionine or methionine hydroxy analog on milk yield and milk composition.
      J. Dairy Sci. 1985; 68: 3350-3354
      10.3168/jds.S0022-0302(85)81245-5
      ). Results of a kinetic study using lactating goats revealed that absorbed Met contributed at least 4% of the phospholipid pool in the plasma and proposed that the lipogenic effect of Met in the liver was one of the mechanisms among others to stimulate milk and milk fat production (
      • Emmanuel B.
      • Kennelly J.J.
      Kinetics of methionine and choline and their incorporation into plasma lipids and milk components in lactating goats.
      J. Dairy Sci. 1984; 67: 1912-1918
      10.3168/jds.S0022-0302(84)81524-6
      ). Consistent with previous studies, graded Met removal resulted in a trend for decreased milk fat yield in the present study (P < 0.1). The milk fat to protein ratio increased linearly with increased Met supply, which indicated a more prominent effect of Met removal on milk protein than on milk fat production.

      Circulating Concentrations and Mammary Metabolism of AA

      Circulating concentrations of an AA are determined by the balance of net appearance and use by body tissues. For an EAA, circulating concentrations generally decrease with decreased postruminal supply (
      • 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.
      J. Dairy Sci. 2000; 83 (10791793): 765-775
      ;
      • Paz H.A.
      • de Veth M.J.
      • Ordway R.S.
      • Kononoff P.J.
      Evaluation of rumen-protected lysine supplementation to lactating dairy cows consuming increasing amounts of distillers dried grains with solubles.
      J. Dairy Sci. 2013; 96 (24035024): 7210-7222
      ;
      • Ying F.
      • Lin X.Y.
      • Ma W.M.
      • Chi H.L.
      • Yan Z.G.
      • Song Y.F.
      • Wang Z.H.
      Metabolic responses to the deficiency of Lys, Arg, Met, or His in the mammary gland of lactating goats.
      Small Rumin. Res. 2013; 113: 219-230
      ;
      • Lin X.Y.
      • Wang J.F.
      • Su P.C.
      • Wang Y.
      • Wang Z.H.
      Lactation responses and mammary amino acid metabolism in lactating goats when complete or Met lacking amino acid mixtures were infused into the jugular vein.
      Small Rumin. Res. 2014; 120: 135-141
      ). In fact, the efficacy of a ruminal protected EAA, such as Lys and Met, can be evaluated based on a standard curve relating postruminal supply to blood concentrations of that specific EAA (
      • Whitehouse N.L.
      • Schwab C.G.
      • Brito A.F.
      The plasma free amino acid dose-response technique: A proposed methodology for determining lysine relative bioavailability of rumen-protected lysine supplements.
      J. Dairy Sci. 2017; 100: 9585-9601
      10.3168/jds.S0022-0302(83)82054-2
      ). Consistent with those expectations, both arterial and venous concentrations of Met decreased linearly with graded Met removal in the present study. Compared with the complete mixture, removing all the Met decreased arterial and venous Met concentrations by 36 and 23%, respectively. The decrease in circulating Met concentrations seemed to be attenuated as reflected by the trend for a quadratic effect for venous concentrations. The quadratic effect on arterial Met was also close to the declared tendency level (P = 0.13).
      Graded Met removal resulted in linear increases in mammary BFMam (Table 9), a phenomenon that has also been observed for graded Lys removal (
      • Guo C.L.
      • Li Y.T.
      • Lin X.Y.
      • Hanigan M.D.
      • Yan Z.G.
      • Hu Z.Y.
      • Hou Q.L.
      • Jiang F.G.
      • Wang Z.H.
      Effects of graded removal of lysine from an intravenously infused amino acid mixture on lactation performance and mammary amino acid metabolism in lactating goats.
      J. Dairy Sci. 2017; 100 (28434735): 4552-4564
      ) and for His (
      • 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.
      J. Dairy Sci. 2000; 83 (10791793): 765-775
      ), Leu (
      • 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.
      J. Dairy Sci. 1996; 79 (9029360): 2217-2224
      ), and Arg deficiencies (
      • Ying F.
      • Lin X.Y.
      • Ma W.M.
      • Chi H.L.
      • Yan Z.G.
      • Song Y.F.
      • Wang Z.H.
      Metabolic responses to the deficiency of Lys, Arg, Met, or His in the mammary gland of lactating goats.
      Small Rumin. Res. 2013; 113: 219-230
      ). Elevated mammary blood flow seems to therefore be a general response to individual EAA deficiency as suggested by
      • 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.
      Can. J. Anim. Sci. 2003; 83: 341-355
      . Similar to
      • Guo C.L.
      • Li Y.T.
      • Lin X.Y.
      • Hanigan M.D.
      • Yan Z.G.
      • Hu Z.Y.
      • Hou Q.L.
      • Jiang F.G.
      • Wang Z.H.
      Effects of graded removal of lysine from an intravenously infused amino acid mixture on lactation performance and mammary amino acid metabolism in lactating goats.
      J. Dairy Sci. 2017; 100 (28434735): 4552-4564
      , NO concentration in the mammary vein plasma was linearly elevated by Met removal in the present study but only at the trend level of significance. If the change in NO is significant, it may at least partially explain the elevated mammary blood flow as NO has been identified as one of the local vasodilatory compounds (
      • Laughlin M.H.
      • Korzick D.H.
      Vascular smooth muscle: Integrator of vasoactive signals during exercise hyperemia.
      Med. Sci. Sports Exerc. 2001; 33 (11194116): 81-91
      ). Such a mechanism helps mitigate the effects of an AA deficiency, which helps ensure sequestration of the necessary substrates to maintain milk production.
      Because blood both delivers and removes metabolites from the tissue, the overall effect on uptake depends on the resulting local tissue concentrations and the affinity of the tissue for the metabolite.
      • 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.
      Br. J. Nutr. 1998; 79 (9505804): 63-78
      discussed the effect of variation in blood flow on tissue removal of a metabolite and pointed out that the effect depended on tissue affinity to that metabolite, which was minor if the affinity was low and was profound if it was moderate or high. Affinity of mammary tissue to individual AA was measured as KClearance in the present study. The combination of decreased arterial concentrations of Met, increased mammary affinity for Met, and increased mammary blood flow as Met supply decreased appeared to completely offset the negative effect of the deficiency in terms of mammary Met uptake. However, despite very similar Met uptake across the treatments, milk protein yield still declined by 30% from the complete infusion to 0 infused Met.
      Although mammary affinity for Met was increased by Met deficiency in the present study, mammary affinity for Lys was not changed by varied Lys supply in a similar study (
      • Guo C.L.
      • Li Y.T.
      • Lin X.Y.
      • Hanigan M.D.
      • Yan Z.G.
      • Hu Z.Y.
      • Hou Q.L.
      • Jiang F.G.
      • Wang Z.H.
      Effects of graded removal of lysine from an intravenously infused amino acid mixture on lactation performance and mammary amino acid metabolism in lactating goats.
      J. Dairy Sci. 2017; 100 (28434735): 4552-4564
      ). As far as we know, Met is an EAA that affects mTORC1 signal pathway in the mammary gland (Table 10) and is predominantly catabolized in the liver (
      • Lapierre H.
      • Berthiaume R.
      • Raggio G.
      • Thivierge M.C.
      • Doepel L.
      • Pacheco D.
      • Dubreuil P.
      • Lobley G.E.
      The route of absorbed nitrogen into milk protein.
      Anim. Sci. 2005; 80: 11-22
      ) but not Lys. Differences in effects on tissue function and metabolism among EAA may therefore contribute to their variations in supply dependence.
      Table 10Effects of graded removal of Met from the AA mixture infused into jugular vein on phosphorylation level [phosphorylation level (P)/total × 100] of downstream proteins in the mechanistic target of rapamycin complex 1 signaling pathway of mammary gland tissue from lactating goats
      ItemPercentage of Met in complete AA mixtureSEP-value
      100%60%30%0%LinearQuadratic
      P-4EBP1/4EBP171.1567.8958.5356.166.2070.0530.96
      P-p70S6k/p70S6k32.9615.8316.699.055.3640.0020.778
      P-eIF2/eIF249.2444.9643.3450.260.1950.9790.741
      Graded Met removal linearly decreased mammary uptake of Thr in the present study (P < 0.05). In fact, Thr was the only measured EAA in which mammary uptake was significantly affected by treatments. The decrease may relate to decreased mammary affinity for Thr as Kclearance for Thr decreased linearly with graded Met removal to a near tendency level (P = 0.108). Mammary uptake of Thr was an issue rarely investigated in previous studies. Arterial infusion of a 5-EAA mixture (Thr, Met, Leu, Phe, and Lys) with or without insulin into lactating cows resulted in substantial variations in mammary uptakes of these AA but were statistically nonsignificant (
      • Metcalf J.A.
      • Sutton J.D.
      • Cockburn J.E.
      • Napper D.J.
      • Beever D.E.
      The influence of insulin and amino acid supply on amino acid uptake by the lactating bovine mammary gland.
      J. Dairy Sci. 1991; 74 (1744271): 3412-3420
      ). The biggest variation was observed for Thr, which was a 230% increase with AA and AA plus insulin infusions relative to that of the control. Removal of Met from a complete AA mixture intravenously infused into lactating goats tended to decrease mammary Kclearance for Thr (P = 0.075), but the decrease in mammary uptake of Thr was nonsignificant (P = 0.188;
      • Lin X.Y.
      • Wang J.F.
      • Su P.C.
      • Wang Y.
      • Wang Z.H.
      Lactation responses and mammary amino acid metabolism in lactating goats when complete or Met lacking amino acid mixtures were infused into the jugular vein.
      Small Rumin. Res. 2014; 120: 135-141
      ).

      Circulating Metabolites, Hormones, and Phosphorylation of Proteins in mTORC1 Pathway

      Graded Met removal elevated circulating urea N and glucose linearly, which indicated increased whole-body catabolism of AA and hepatic gluconeogenesis, although the reduced use of glucose for lactose synthesis might also have contributed to the elevated circulating glucose. Elevation in circulating glucose was also observed in our previous Lys removal experiment (
      • Guo C.L.
      • Li Y.T.
      • Lin X.Y.
      • Hanigan M.D.
      • Yan Z.G.
      • Hu Z.Y.
      • Hou Q.L.
      • Jiang F.G.
      • Wang Z.H.
      Effects of graded removal of lysine from an intravenously infused amino acid mixture on lactation performance and mammary amino acid metabolism in lactating goats.
      J. Dairy Sci. 2017; 100 (28434735): 4552-4564
      ) but not circulating urea N. Circulating total protein was linearly decreased in the Lys removal experiment, which was not observed in the present study. For the 4 hormones measured, Met removal only decreased circulating GH to the declared tendency level. In our previous study, graded Lys removal linearly increased circulating glucagon and increased circulating insulin to a near tendency level (P = 0.153) but had no significant effect on circulating GH. The above results indicated differed whole-body responses to Met and Lys deficiency due to their differences in tissue function and metabolism aside from their common roles in protein synthesis.
      Amino acid availability may also regulate milk protein synthesis at the cellular level through the mTORC1 or GCN signaling pathways. Individual AA may promote milk protein synthesis by repressing the inhibitory activity of 4EBP1, by stimulating eIF2, and possibly by enhancing ribosomal activity through activation of ribosomal protein S6 (
      • Arriola Apelo S.I.
      • Knapp J.R.
      • Hanigan M.D.
      Invited review: Current representation and future trends of predicting amino acid utilization in the lactating dairy cow.
      J. Dairy Sci. 2014; 97 (24767883): 4000-4017
      ). Graded Met removal reduced phosphorylation status of 4EBP1 and that of p70S6k linearly, both of which would downregulate milk protein synthesis and explained the decreased milk protein yields by graded Met removal without a concomitant decrease in mammary uptakes of Met. Similar responses in the mTORC1 signaling pathway were not observed in our previous Lys experiment (
      • Guo C.L.
      • Li Y.T.
      • Lin X.Y.
      • Hanigan M.D.
      • Yan Z.G.
      • Hu Z.Y.
      • Hou Q.L.
      • Jiang F.G.
      • Wang Z.H.
      Effects of graded removal of lysine from an intravenously infused amino acid mixture on lactation performance and mammary amino acid metabolism in lactating goats.
      J. Dairy Sci. 2017; 100 (28434735): 4552-4564
      ).
      Given the lack of changes in uptake of EAA or hormones that might regulate milk protein synthesis, it is difficult to explain the observed changes in cell signaling and the decline in milk protein output. The data suggest that the animals were able to completely mitigate the Met shortage at the tissue level in terms of Met uptake, yet changes in cell signaling and milk protein production were consistent with a 30% change in supply and signaling. No changes occurred in measured hormone concentrations that would help to explain this dichotomy. One would certainly expect a decline in milk protein production given a significant deficiency in Met supply, but the mechanism of action remains difficult to identify.

      CONCLUSIONS

      Graded removal of Met from an intravenously infused AA mixture in to lactating goats linearly decreased milk protein yield. The decrease was clearly not a result of limited Met availability as mammary Met uptake was similar across treatments even when all the Met was removed from the infused AA mixture. Comparison between results of the present and our previous graded Lys removal experiment revealed that whole-body responses, including circulating urea N, total protein, and pancreatic hormones, to deficiency of the 2 AA were different. Intracellular mTORC1 signaling pathway was affected by short-term Met deficiency and might play a part in decreased milk protein synthesis as indicated by the varied phosphorylation status of 4EBP1 and p70S6k.

      ACKNOWLEDGMENTS

      The work is funded by National Natural Science Foundation of China (Beijing) under project number 31372340 and by China Agricultural Research System (Beijing) under the project CARS-37.

      REFERENCES

        • AFRC
        Energy and Protein Requirements of Ruminants.
        CAB International, Wallingford, UK1993
        View in Article
        • Appuhamy J.A.
        • Knapp J.R.
        • Becvar O.
        • Escobar J.
        • Hanigan M.D.
        Effects of jugular-infused lysine, methionine, and branched-chain amino acids on milk protein synthesis in high-producing dairy cows.
        J. Dairy Sci. 2011; 94 (21426986): 1952-1960
        View in Article
        • Appuhamy J.A.D.R.N.
        Regulatory roles of essential amino acids, energy, and insulin in mammary cell protein synthesis.
        (PhD Diss.) Animal Sciences, Virginia Polytechnic Institute and State University, Blacksburg2010
        View in Article
        • Arriola Apelo S.I.
        • Knapp J.R.
        • Hanigan M.D.
        Invited review: Current representation and future trends of predicting amino acid utilization in the lactating dairy cow.
        J. Dairy Sci. 2014; 97 (24767883): 4000-4017
        View in Article
        • Baker D.H.
        Advances in amino acid nutrition and metabolism of swine and poultry.
        in: Kornegay E.T. Nutrient Management of Food Animals to Enhance and Protect the Environment. CRC Press, Boca Raton, FL1996: 41-52
        View in Article
        • 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.
        J. Dairy Sci. 1996; 79 (9029360): 2217-2224
        View in Article
        • 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.
        J. Dairy Sci. 2000; 83 (10791793): 765-775
        View in Article
        • Bequette B.J.
        • Kyle C.
        • Crompton L.
        • Buchan V.
        • Hanigan M.
        Insulin regulates milk production and mammary gland and hind-leg amino acid fluxes and blood flow in lactating goats.
        J. Dairy Sci. 2001; 84 (11210039): 241-255
        View in Article
        • Berthiaume R.
        • Dubreuil P.
        • Stevenson M.
        • McBride B.W.
        • Lapierre H.
        Intestinal disappearance, mesenteric and portal appearance of amino acids in dairy cows fed ruminally protected methionine.
        J. Dairy Sci. 2001; 84 (11210033): 194-203
        View in Article
        • Berthiaume R.
        • Thivierge M.C.
        • Lobley G.E.
        • Dubreuil P.
        • Babkine M.
        • Lapierre H.
        Effect of a jugular infusion of essential amino acids on splanchnic metabolism in dairy cows fed a protein deficient diet.
        J. Dairy Sci. 2002; 85 (Abstr.): 72-73
        View in Article
        • 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.
        Can. J. Anim. Sci. 2003; 83: 341-355
        View in Article
        • Chen Z.H.
        • Broderick G.A.
        • Luchini N.D.
        • Sloan B.K.
        • Devillard E.
        Effect of feeding different sources of rumen-protected methionine on milk production and N-utilization in lactating dairy cows.
        J. Dairy Sci. 2011; 94: 1978-1988
        10.3168/jds.2010-3578
        View in Article
        • Cho J.
        • Overton T.R.
        • Schwab C.G.
        • Tauer L.W.
        Determining the amount of rumen-protected methionine supplement that corresponds to the optimal levels of methionine in metabolizable protein for maximizing milk protein production and profit on dairy farms.
        J. Dairy Sci. 2007; 90: 4908-4916
        10.3168/jds.2007-0314
        View in Article
        • Donkin S.S.
        • Varga G.A.
        • Sweeney T.F.
        • Muller L.D.
        Rumen-protected methionine and lysine: Effects on animal performance, milk protein yield, and physiological measures.
        J. Dairy Sci. 1989; 72: 1484-1491
        10.3168/jds.S0022-0302(89)79258-4
        View in Article
        • Emmanuel B.
        • Kennelly J.J.
        Kinetics of methionine and choline and their incorporation into plasma lipids and milk components in lactating goats.
        J. Dairy Sci. 1984; 67: 1912-1918
        10.3168/jds.S0022-0302(84)81524-6
        View in Article
        • Guinard J.
        • Rulquin H.
        Effect of graded amount of duodenal infusions of methionine on the mammary uptake of major milk precursors in dairy cows.
        J. Dairy Sci. 1995; 78 (8598404): 2196-2207
        View in Article
        • Guo C.L.
        • Li Y.T.
        • Lin X.Y.
        • Hanigan M.D.
        • Yan Z.G.
        • Hu Z.Y.
        • Hou Q.L.
        • Jiang F.G.
        • Wang Z.H.
        Effects of graded removal of lysine from an intravenously infused amino acid mixture on lactation performance and mammary amino acid metabolism in lactating goats.
        J. Dairy Sci. 2017; 100 (28434735): 4552-4564
        View in Article
        • Güttler F.
        • Kühl C.
        • Pedersen L.
        • Påby P.
        Effects of oral phenylalanine load on plasma glucagon, insulin, AA and glucose concentrations in man.
        Scand. J. Clin. Lab. Invest. 1978; 38 (663549): 255-260
        View in Article
        • Hanigan M.D.
        • Cant J.P.
        • Weakley D.C.
        • Beckett J.L.
        An evaluation of post absorptive protein and amino acid metabolism in the lactating dairy cow.
        J. Dairy Sci. 1998; 81 (9891282): 3385-3401
        View in Article
        • Hanigan M.D.
        • Reynolds C.
        • Humphries D.
        • Lupoli B.
        • Sutton J.
        A model of net amino acid absorption and utilization by the portal-drained viscera of the lactating dairy cow.
        J. Dairy Sci. 2004; 87 (15545389): 4247-4268
        View in Article
        • Hanigan M.D.
        • France J.
        • Crompton L.A.
        • Bequette B.J.
        Evaluation of a representation of the limiting amino acid theory for milk protein synthesis.
        in: McNamara J.P. France J. Beever D.E. Modelling Nutrient Utilization in Farm Animals. CABI, Wallingford, UK2000: 127-144
        View in Article
        • 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.
        Br. J. Nutr. 1998; 79 (9505804): 63-78
        View in Article
        • Hristov A.N.
        • Price W.J.
        • Shafii B.
        A meta-analysis examining the relationship among dietary factors, dry matter intake, and milk and milk protein yield in dairy cows.
        J. Dairy Sci. 2004; 87 (15328233): 2184-2196
        View in Article
        • Kim S.G.
        • Buel G.R.
        • Blenis J.
        Nutrient regulation of the mTOR complex 1 signaling pathway.
        Mol. Cells. 2013; 35 (23694989): 463-473
        View in Article
        • Lapierre H.
        • Berthiaume R.
        • Raggio G.
        • Thivierge M.C.
        • Doepel L.
        • Pacheco D.
        • Dubreuil P.
        • Lobley G.E.
        The route of absorbed nitrogen into milk protein.
        Anim. Sci. 2005; 80: 11-22
        View in Article
        • Lapierre H.
        • Lobley G.E.
        • Ouellet D.R.
        • Doepel L.
        • Pacheco D.
        Amino acid requirements for lactating dairy cows: Reconciling predictive models and biology.
        in: Proc. Cornell Nutrition Conference for Feed Manufacturers. Department of Animal Science, Cornell University, Ithaca, NY2007: 39-59
        View in Article
        • Laughlin M.H.
        • Korzick D.H.
        Vascular smooth muscle: Integrator of vasoactive signals during exercise hyperemia.
        Med. Sci. Sports Exerc. 2001; 33 (11194116): 81-91
        View in Article
        • Lee C.
        • Hristov A.N.
        • Cassidy T.W.
        • Heyler K.S.
        • Lapierre H.
        • Varga G.A.
        • de Veth M.J.
        • Patton R.A.
        • Parys C.
        Rumen-protected lysine, methionine, and histidine increase milk protein yield in dairy cows fed a metabolizable protein-deficient diet.
        J. Dairy Sci. 2012; 95: 6042-6056
        10.3168/jds.2012-5581
        View in Article
        • Lin X.Y.
        • Wang J.F.
        • Su P.C.
        • Wang Y.
        • Wang Z.H.
        Lactation responses and mammary amino acid metabolism in lactating goats when complete or Met lacking amino acid mixtures were infused into the jugular vein.
        Small Rumin. Res. 2014; 120: 135-141
        View in Article
        • Liu G.M.
        • Hanigan M.D.
        • Lin X.Y.
        • Zhao K.
        • Jiang F.G.
        • White R.R.
        • Wang Y.
        • Hu Z.Y.
        • Wang Z.H.
        Methionine, leucine, isoleucine, or threonine effects on mammary cell signaling and pup growth in lactating mice.
        J. Dairy Sci. 2017; 100 (28237591): 4038-4050
        View in Article
        • Lundquist R.G.
        • Otterby D.E.
        • Linn J.G.
        Influence of three concentrations of DL-methionine or methionine hydroxy analog on milk yield and milk composition.
        J. Dairy Sci. 1985; 68: 3350-3354
        10.3168/jds.S0022-0302(85)81245-5
        View in Article
        • Mackle T.R.
        • Dwyer D.A.
        • Ingvartsen K.L.
        • Chouinard P.Y.
        • Ross D.A.
        • Bauman D.E.
        Effects of insulin and postruminal supply of protein on use of amino acids by the mammary gland for milk protein synthesis.
        J. Dairy Sci. 2000; 83 (10659969): 93-105
        View in Article
        • Metcalf J.A.
        • Sutton J.D.
        • Cockburn J.E.
        • Napper D.J.
        • Beever D.E.
        The influence of insulin and amino acid supply on amino acid uptake by the lactating bovine mammary gland.
        J. Dairy Sci. 1991; 74 (1744271): 3412-3420
        View in Article
        • Misciattelli L.
        • Kristensen V.F.
        • Vestergaard M.
        • Weisbjerg M.R.
        • Sejrsen K.
        • Hvelplund T.
        Milk production, nutrient utilization, and endocrine responses to increased postruminal lysine and methionine supply in dairy cows.
        J. Dairy Sci. 2003; 86: 275-286
        10.3168/jds.S0022-0302(03)73606-6
        View in Article
        • Nahm K.H.
        Efficient feed nutrient utilization to reduce pollutants in poultry and swine manure.
        Crit. Rev. Environ. Sci. Technol. 2002; 32: 1-16
        View in Article
        • Noftsger S.
        • St-Pierre N.R.
        Supplementation of methionine and selection of highly digestible rumen undegradable protein to improve nitrogen efficiency for milk production.
        J. Dairy Sci. 2003; 86: 958-969
        10.3168/jds.S0022-0302(03)73679-0
        View in Article
        • NRC
        Nutrient Requirements of Dairy Cattle.
        7th rev. ed. Natl. Acad. Press, Washington, DC2001
        View in Article
        • Paz H.A.
        • de Veth M.J.
        • Ordway R.S.
        • Kononoff P.J.
        Evaluation of rumen-protected lysine supplementation to lactating dairy cows consuming increasing amounts of distillers dried grains with solubles.
        J. Dairy Sci. 2013; 96 (24035024): 7210-7222
        View in Article
        • Rogers J.A.
        • Krishnamoorthy U.
        • Sniffen C.J.
        Plasma amino acids and milk protein production by cows fed rumen-protected methionine and lysine.
        J. Dairy Sci. 1987; 70: 789-798
        10.3168/jds.S0022-0302(87)80075-9
        View in Article
        • Stone K.P.
        • Wanders D.
        • Orgeron M.
        • Cortez C.C.
        • Gettys T.W.
        Mechanisms of increased in vivo insulin sensitivity by dietary methionine restriction in mice.
        Diabetes. 2014; 63 (24947368): 3721-3733
        View in Article
        • Whitehouse N.L.
        • Schwab C.G.
        • Brito A.F.
        The plasma free amino acid dose-response technique: A proposed methodology for determining lysine relative bioavailability of rumen-protected lysine supplements.
        J. Dairy Sci. 2017; 100: 9585-9601
        10.3168/jds.S0022-0302(83)82054-2
        View in Article
        • Wray-Cahen D.
        • Metcalf J.
        • Backwell F.
        • Bequette B.
        • Brown D.
        • Sutton J.
        • Lobley G.
        Hepatic response to increased exogenous supply of plasma amino acids by infusion into the mesenteric vein of Holstein-Friesian cows in late gestation.
        Br. J. Nutr. 1997; 78 (9497443): 913-930
        View in Article
        • Ying F.
        • Lin X.Y.
        • Ma W.M.
        • Chi H.L.
        • Yan Z.G.
        • Song Y.F.
        • Wang Z.H.
        Metabolic responses to the deficiency of Lys, Arg, Met, or His in the mammary gland of lactating goats.
        Small Rumin. Res. 2013; 113: 219-230
        View in Article
        • Zhou Z.
        • Vailati-Riboni M.
        • Trevisi E.
        • Drackley J.K.
        • Luchini D.N.
        • Loor J.J.
        Better postpartal performance in dairy cows supplemented with rumen-protected methionine compared with choline during the peripartal period.
        J. Dairy Sci. 2016; 99: 1-17
        10.3168/jds.S0022-0302(83)82054-2
        View in Article

      Article info

      Publication history

      Published online: March 01, 2019
      Accepted: December 20, 2018
      Received: September 4, 2018

      Identification

      DOI: https://doi.org/10.3168/jds.2018-15643

      Copyright

      © 2019 American Dairy Science Association®.

      User license

      Elsevier user license |
      How you can reuse Information Icon
      Elsevier user license

      Permitted

      For non-commercial purposes:

      • Read, print & download
      • Text & data mine
      • Translate the article

      Not Permitted

      • Reuse portions or extracts from the article in other works
      • Redistribute or republish the final article
      • Sell or re-use for commercial purposes

      Elsevier's open access license policy

      ScienceDirect

      Access this article on ScienceDirect

      The content on this site is intended for healthcare professionals.


      We use cookies to help provide and enhance our service and tailor content. To update your cookie settings, please visit the Cookie Preference Center for this site.
      Copyright © 2023 Elsevier Inc. except certain content provided by third parties.


      RELX