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Published December 8, 2014 Excess amino acid supply improves methionine and leucine utilization by growing steers1 M. S. Awawdeh,* E. C. Titgemeyer,*2 G. F. Schroeder,* and D. P. Gnad† *Department of Animal Sciences and Industry and †Department of Clinical Sciences, Kansas State University, Manhattan 66506-1600 ABSTRACT: In 2 experiments, 6 ruminally cannulated Holstein steers (205 ± 23 and 161 ± 14 kg initial BW in Exp. 1 and 2, respectively) housed in metabolism crates were used in 6 × 6 Latin squares to study the effects of excess AA supply on Met (Exp. 1) and Leu (Exp. 2) use. All steers received a diet based on soybean hulls (DMI = 2.66 and 2.45 kg/d in Exp. 1 and 2, respectively); ruminal infusions of 200 g of acetate/d, 200 g of propionate/d, and 50 g of butyrate/d, as well as abomasal infusion of 300 g of glucose/d to provide energy without increasing the microbial protein supply; and abomasal infusions of a mixture of all essential AA except Met (Exp. 1) or Leu (Exp. 2). Periods were 6 d, with 2-d adaptations and 4 d to collect N balance data. All treatments were abomasally infused. In Exp. 1, treatments were arranged as a 2 × 3 factorial, with 2 amounts of L-Met (0 or 4 g/d) and 3 AA supplements (no additional AA, control; 100 g/d of nonessential AA + 100 g/d of essential AA, NEAA + EAA; and 200 g/d of essential AA, EAA). Supplemental Met increased (P < 0.01) retained N and decreased (P < 0.01) urinary N and urinary urea N. Retained N increased (P < 0.01) with NEAA + EAA only when 4 g/d of Met was provided, but it increased (P < 0.01) with EAA with or without supplemental Met. Both AA treatments increased (P < 0.01) plasma urea and serum insulin. Plasma glucose decreased (P = 0.03) with supplemental Met. In Exp. 2, treatments were arranged as a 2 × 3 factorial with 2 amounts of L-Leu (0 or 4 g/d) and 3 AA supplements (control, NEAA + EAA, and EAA). Supplemental Leu increased (P < 0.01) retained N and decreased (P < 0.01) urinary N and urinary urea N. Both AA treatments increased (P < 0.01) retained N, and they also increased (P < 0.01) urinary N, urinary urea N, and plasma urea. Serum insulin increased (P = 0.06) with supplemental Leu and tended (P = 0.10) to increase with both AA treatments. Supplementation with excess AA improved Met and Leu use for protein deposition by growing cattle. Key words: amino acid, cattle, growth, leucine, methionine, utilization 2006 American Society of Animal Science. All rights reserved. INTRODUCTION In vivo (Lobley et al., 1995) and in vitro (Mutsvangwa et al., 1996, 1999) studies suggested that metabolic ammonia (NH3) loading might lead to inefficient use of dietary AA by metabolically consuming AA to provide α-amino N to support ureagenesis. We recently observed no negative effects of NH3 loading on protein deposition when Met, Leu, or His limited cattle performance (Awawdeh et al., 2004, 2005; McCuistion et al., 2004). Excess dietary N had different effects on protein 1 Contribution No. 06-39-J from the Kansas Agric. Exp. Stn., Manhattan. This research was supported by NRI Competitive Grants Program/CSREES/USDA, Award No. 2003-35206-12837. 2 Corresponding author: [email protected] Received September 28, 2005. Accepted February 9, 2006. J. Anim. Sci. 2006. 84:1801–1810 doi:10.2527/jas.2005-557 deposition, depending on the N source. For example, supplementation with essential AA had no significant effects on His use, but supplementation with a mixture of essential and nonessential AA improved His use in cattle (McCuistion et al., 2004). We have demonstrated that Met and Leu are used by growing cattle with efficiencies less than the NRC (1996) values (Awawdeh et al., 2004, 2005), and that efficiency of Leu use depends on the animal’s nutritional status (Awawdeh et al., 2005). Our long-term objective is to evaluate use efficiencies of individual AA under different nutritional conditions. We have measured the use efficiency for Met, Leu, and His under NH3 loading (Awawdeh et al., 2004, 2005; McCuistion et al., 2004), for Met with supplemental energy (Schroeder et al., 2006a), and for His with excess AA supply (McCuistion et al., 2004). Because Met, Leu, and His are metabolized differently throughout the body, excess AA could have differ- 1801 1802 Awawdeh et al. Table 2. Ruminal and abomasal infusates (Exp. 1 and 2) Table 1. Diet composition (Exp. 1 and 2) Item % of DM Item Ingredient Pelleted soybean hulls Wheat straw Cane molasses Dicalcium phosphate Sodium bicarbonate Calcium carbonate Urea Magnesium oxide Trace mineralized salt1 Vitamin premix2 Sulfur Bovatec-683 83.4 7.4 3.7 2.0 1.2 1.0 0.3 0.4 0.2 0.2 0.1 + Nutrient OM N 89.6 2.1 1 Composition (g/kg, minimum guarantee): NaCl (960 to 990), Mn (≥ 2.4), Fe (≥ 2.4), Mg (≥ 0.5), Cu (≥ 0.32), Zn (≥ 0.32), I (≥ 0.07), and Co (≥ 0.04). 2 Provided 4,410 IU of vitamin A, 2,205 IU of vitamin D, and 45 IU of vitamin E per kilogram of diet DM. 3 Supplied 33 mg of lasalocid per kilogram of diet DM. ent effects on their use by growing cattle. For example, the first committed step in Met catabolism is cystathionine synthesis, a process that competes with methylation for homocysteine; His catabolism is regulated in part by supply of AA other than His; Leu is catabolized throughout the body, rather than principally in the liver, and the initial step is transamination. Our objective was to examine effects of excess AA on the wholebody protein deposition when Met or Leu was the most limiting AA. MATERIALS AND METHODS The Kansas State University Institutional Animal Care and Use Committee approved all procedures involving animals in this study. In 2 experiments, 6 ruminally cannulated Holstein steers (205 ± 23 and 161 ± 14 kg initial BW in Exp. 1 and 2, respectively) fitted with ruminal and abomasal infusion lines were used in 6 × 6 Latin squares to study the effects of excess AA on Met (Exp. 1) and Leu (Exp. 2) use. Steers were housed in individual metabolism crates in a temperature-controlled room (21°C) under continuous lighting. Before initiation of each experiment, steers were adapted to the basal diet (Table 1) for 2 wk and to ruminal and abomasal infusions for 5 d. All steers had free access to water and received the same basal diet in equal proportions at 12-h intervals, with DMI averaging 2.66 and 2.45 kg/d in Exp. 1 and 2, respectively. The basal diet, which was characterized for metabolizable AA supply by Campbell et al. (1997), was formulated to provide adequate ruminally degraded protein, but small amounts of metabolizable AA. All steers received continuous ruminal infusions of 200 g of acetate/ Control NEAA + EAA 0 or 4 10 20 0 or 4 10 20 10 10 10 5 10 10 100 50 — — — — 300 60 30 34 0 or 4 15 35 0 or 4 15 40 20 25 20 10 20 20 130 60 15 25 10 10 300 90 42 17 Abomasal infusate L-Met (Exp. 1) (Exp. 2) L-Leu (Exp. 1) (Exp. 2) 1 L-HisⴢHClⴢH2O (74.0%) 2 L-LysⴢHCl (feed grade; 78.8%) 3 L-Thr (feed grade; 98%) L-Phe L-Arg 4 L-Trp (feed grade; 98%) L-Ile L-Val L-Glu Glycine L-Ala L-Asp L-Pro L-Ser Dextrose 6 M HCl NaOH NaCl EAA g/d 0 or 4 20 50 0 or 4 20 60 30 40 30 15 30 30 100 50 — — — — 300 120 54 — mg/d Folic acid PyridoxineⴢHCl Cyanocobalamin 10 10 0.1 10 10 0.1 g/d Ruminal infusate Acetic acid Propionic acid Butyric acid 10 10 0.1 200 200 50 200 200 50 200 200 50 1 Provided 7.4, 11.1, and 14.8 g of His/d for control, nonessential AA + essential AA (NEAA + EAA), and EAA, respectively. 2 Provided 15.8, 31.5, and 47.3 g of Lys/d for control, NEAA + EAA, and EAA, respectively. 3 Provided 9.8, 19.6, and 29.4 g of Thr/d for control, NEAA + EAA, and EAA, respectively. 4 Provided 4.9, 9.8, and 14.7 g of Trp/d for control, NEAA + EAA, and EAA, respectively. d, 200 g of propionate/d, and 50 g of butyrate/d as well as abomasal infusions of 300 g of glucose/d to supply additional energy without increasing the microbial protein supply. To ensure that the most limiting AA for N retention was Met in Exp. 1 and Leu in Exp. 2, all steers received continuous abomasal infusions of an AA mixture (Table 2; control group), as described by Greenwood and Titgemeyer (2000), that supplied nonessential and all essential AA, except for Met in Exp. 1 and Leu in Exp. 2. In Exp. 1, treatments (Table 2) were arranged as a 2 × 3 factorial, with 2 amounts of L-Met (0 or 4 g/d) and 3 AA supplements (Table 2), including no additional AA (control), 100 g/d of nonessential AA + 100 g/d of essential AA (NEAA + EAA), and 200 g/d of essential AA (EAA). All treatments were delivered to the abomasum via continuous infusion. To ensure that the steers were able to respond to supplemental Met, the 4 g/d of supplemental Met was less than the steers’ require- Amino acid utilization by cattle ments for maximal N retention under our experimental conditions (Campbell et al., 1997). The EAA and NEAA + EAA treatments were estimated to increase the metabolizable AA supply above that of the control group by approximately 40%. The 2 groups of AA (NEAA + EAA and EAA) were selected to examine if the effects of excess AA supply were dependent on AA type (essential vs. nonessential) or simply on the amount of N provided. Each experimental period lasted 6 d, with 2 d for adaptation to treatment and 4 d for total fecal and urinary collections. Short adaptation periods are adequate because cattle rapidly adapt to changes in nutrients supplied postruminally (Moloney et al., 1998), and 2-d adaptations have been validated for our experimental model (Schroeder et al., 2006b). Abomasal infusate for the control treatment was prepared by dissolving the branched-chain AA (L-Val, LLeu, and L-Ile) in 1 kg of water containing 60 g of 6 M HCl. Once the branched-chain AA were dissolved, the remaining AA, except L-Glu, were added to the mixture. Glutamate was dissolved separately in 500 g of water containing 30 g of NaOH. After all AA were dissolved, the 2 solutions of AA were mixed together, 300 g of glucose was added, and water was added to bring the total weight of the daily infusate to 4 kg. The EAA and NEAA + EAA treatments were prepared by replacing 2 kg of water from the control infusate with solutions containing AA. For EAA treatment, branched-chain AA were dissolved in 1.7 kg of water containing 60 g of 6 M HCl. After the branched-chain AA were dissolved, the remaining AA (L-Arg, L-His, LLys, L-Phe, L-Thr, and L-Trp) were added to the mixture, and water was added to bring the final weight to 2 kg. The NEAA + EAA treatment was prepared by replacing 2 kg of water from the control infusate with 1 kg of the EAA solution and 1 kg of the NEAA solution. The NEAA solution was prepared by dissolving L-Glu and L-Asp with 800 g of water containing 11 g of NaOH. Once those dissolved, the remaining AA (Gly, L-Ala, L-Pro, and L-Ser) were added to the solution, and water was added to bring the final weight to 2 kg. The AA treatments were balanced for Na and Cl (from HCl and NaOH used to prepare the AA solutions) by adding 34 g of NaCl to the control, 17 g of NaCl to NEAA + EAA, and 24 g of NaOH to EAA. PyridoxineⴢHCl (10 mg), folic acid (10 mg), and cyanocobalamin (100 g) were added to the abomasal infusate because steers maintained under our experimental conditions were deficient in one or more of these vitamins (Lambert et al., 2004). Methionine was dissolved separately in water and added to the mixture according to treatment (0 or 4 g). Ruminal infusates for each steer were prepared by mixing 200 g of acetic acid/d, 200 g of propionic acid/d, 50 g of butyric acid/d, and 3.55 kg of water/d. Infusion lines of flexible polyvinylchloride tubing (2.4-mm i.d.) were placed in the rumen and abomasum through the ruminal cannula. A perforated vial was attached to the 1803 end of the ruminal infusion lines to avoid direct infusion of VFA onto the ruminal wall. Rubber flanges (8-cm diam.) were attached to the end of the abomasal infusion lines to ensure that they remained in the abomasum. Solutions were continuously infused into the rumen and abomasum by using a peristaltic pump. Representative samples of the basal diet for each period were collected daily and stored (−20°C) for later analysis. Orts, if any, were collected on d 2 through 5, composited, and stored (−20°C) for later analysis. Feces and urine for each steer were collected from d 3 through 6 of each period and were weighed to determine output. Urine was collected in buckets containing 300 mL of 6 M HCl to prevent NH3 loss. Representative samples of feces (10%) and urine (1%) were saved, composited by period, and stored (−20°C) for later analysis. Three observations were not obtained in Exp. 1 due to failures in the collection of excreta. Six observations were not obtained in the original design in Exp. 2 as a result of failure to collect excreta. Thus, in Exp. 2, the Latin square was modified by adding an additional period (observations were collected from 5 steers), with a treatment distribution to compensate for the missing data, to increase the final number of observations to 35. Samples of the diet, orts, and feces were analyzed for DM (105°C in a forced-air oven for 24 h) and OM (weight loss upon ashing at 450°C for 8 h) to calculate digestibilities. Composite samples of the diet, orts, wet feces, and urine were analyzed for N by using a Leco FP 2000 Nitrogen Analyzer (Leco Corporation, St. Joseph, MI) to calculate N retention. Jugular blood samples were collected 4 h after the morning feeding on the last day of each period. Blood was collected into vacuum tubes (Becton Dickinson, Franklin Lakes, NJ) containing sodium heparin, immediately chilled on ice, and centrifuged for 20 min at 1,000 × g to obtain plasma. Blood also was collected into vacuum tubes without additives, allowed to clot for 30 min at room temperature, and centrifuged for 20 min at 1,000 × g to obtain serum. Samples were stored (−20°C) for later analysis of plasma glucose, urea, and AA, and of serum insulin and IGF-I. Plasma glucose concentrations were measured according to methods of Gochman and Schmitz (1972). Plasma and urinary urea were measured according to the method of Marsh et al. (1965), and urinary and ruminal fluid NH3 concentrations were measured by the method of Broderick and Kang (1980). Plasma AA were measured by gas chromatography with a commercial kit (EZ:faast, Phenomenex, Torrance, CA). Insulin was measured with an insulin RIA kit (intraassay CV of 2.0% and sensitivity of 0.024 ng/mL; DSL-1600, Diagnostic Systems Laboratories, Webster, TX), and IGF-I was measured with an active IGF-I coated-tube, 2-site immunoradiometric assay kit (intraassay CV of 1.0% and sensitivity of 5.0 ng/mL; DSL5600, Diagnostic Systems Laboratories). Data were analyzed statistically as a Latin square by using the MIXED procedure of SAS, Release 8.1 1804 Awawdeh et al. Table 3. Effect of supplemental Met and AA on nitrogen balance and diet digestibility of growing steers (Exp. 1)1 P value No Met Item n 4 g of Met/d Control NEAA + EAA EAA Control NEAA + EAA EAA SEM2 Met Control vs. AA EAA vs. EAA + NEAA Met × (Control vs. AA) Met × (EAA vs. EAA + NEAA) 6 4 5 6 6 6 Nitrogen, g/d Dietary Infused Total intake Fecal Urinary Urea Ammonia Retained 57.9 34.6 92.5 20.2 49.9 37.0 1.7 22.3 56.9 61.1 118.0 19.4 77.9 60.9 3.9 21.6 55.6 63.2 118.8 18.6 70.8 56.8 6.3 29.8 56.9 35.0 91.9 19.1 45.3 33.4 1.5 27.5 56.0 61.5 117.6 19.4 60.6 47.2 4.0 37.5 57.2 63.5 120.8 18.6 65.7 53.2 2.0 36.4 1.3 — 1.3 1.0 2.3 2.1 1.2 2.6 0.91 0.19 0.97 0.35 0.18 0.67 0.66 <0.01 <0.01 0.10 <0.01 <0.01 0.49 <0.01 <0.01 0.01 <0.01 0.03 0.48 0.66 0.64 0.83 0.17 0.35 0.51 0.09 0.14 0.31 0.15 0.18 0.97 0.02 0.02 0.06 0.08 Diet digestibility, % DM OM 75.2 78.0 75.6 77.9 75.5 77.7 75.0 77.0 75.2 77.9 75.1 77.5 1.1 1.2 0.74 0.70 0.80 0.81 0.94 0.81 0.88 0.68 0.99 0.98 1 NEAA + EAA = 100 g/d nonessential + 100 g/d essential AA; EAA = 200 g/d essential AA. For n = 6. 2 (SAS Inst. Inc., Cary, NC). The model contained the effects of Met, AA, Met × AA, and period. Steer was included as a random variable. Treatment means were computed using the LSMEANS option. Orthogonal contrasts were used to evaluate the effects of AA treatments and their interactions with Met: 1) control vs. average of EAA and NEAA + EAA, and 2) EAA vs. NEAA + EAA. Pairwise t-tests among least squares means were used to derive P values for comparisons that could not be easily described by the contrasts. In Exp. 2, experimental housing, periods, diet, basal infusions, sample collection, laboratory analyses, and statistical analysis were the same as in Exp. 1, except for Leu being the most limiting AA instead of Met. Treatments were arranged as a 2 × 3 factorial, with 2 amounts of L-Leu (0 or 4 g/d) and 3 AA supplements (control, NEAA + EAA, and EAA). The AA treatments were the same as in Exp. 1, except for Met replacing Leu (Table 2). On the basis of data from Awawdeh et al. (2005), 4 g/d of Leu was less than the steers’ requirements under our experimental conditions. RESULTS AND DISCUSSION Experiment 1 Diet digestibilities and N retention data in response to supplemental Met and AA are presented in Table 3. Diet DM and OM digestibilities were not altered (P ≥ 0.68) by treatments and averaged 75.3 and 77.7%, respectively. Fecal N was not affected (P ≥ 0.48) by treatments. Supplementation with 4 g of Met/d increased (P < 0.01) retained N from 24.6 to 33.8 g/d, as a result of decreased (P < 0.01) urinary N excretion, indicating that Met was indeed the limiting AA in our experiment. Similar responses to Met have been consistently ob- served with our experimental model (Campbell et al., 1996, 1997; Awawdeh et al., 2004). Also, supplemental Met decreased (P < 0.01) urinary urea N excretion. The decreases in urinary N and urinary urea N, and the increases in retained N in response to supplemental Met were or tended to be greater for the NEAA + EAA treatment than for the control or the EAA treatment [Met × (EAA vs. NEAA + EAA) interaction; P = 0.02 for urinary N, P = 0.08 for N retention]. Both AA treatments led to increases (P < 0.01) in urinary urea N that were closely proportional to the increases in total urinary N. Both AA treatments increased (P = 0.01) urinary NH3-N. The NEAA + EAA treatment increased (P < 0.01) urinary N less with 4 g of supplemental Met/ d than with no supplemental Met (increases of 15.3 vs. 28.0 g/d, respectively). The EAA treatment increased urinary N the same amount with or without supplemental Met (increases of 20.9 and 20.4 g/d, respectively). The increases in N intake in response to the EAA treatment exceeded the increases in urinary N, resulting in N retention being increased 8.2 g/d by the EAA treatment. The increases in retained N in response to the EAA and NEAA + EAA treatments imply that both AA treatments improved the efficiency of Met use because they increased N retention with the same amount of supplemental Met. This response could be attributed to the energy supplied by the AA treatments (Schroeder et al., 2006a). Our observations that increased AA supply improved, or had no negative impact on, protein deposition when Met limited N retention is in agreement with the finding of McCuistion et al. (2004) that supplying excess AA had either no negative effect or a positive effect on protein deposition when His limited steers’ performance. Although it could be argued that our AA treatments improved N retention by supplying a colimiting AA 1805 Amino acid utilization by cattle Table 4. Effect of supplemental Met and AA on serum hormone and plasma metabolite concentrations of growing steers (Exp. 1)1 P value No Met Item n 4 g of Met/d Control NEAA + EAA EAA 6 5 5 Serum Insulin IGF-I Met × (EAA vs. NEAA + EAA) SEM2 Met Control vs. AA 1.02 174 0.2 21 0.75 0.45 <0.01 0.99 0.18 0.72 0.54 0.40 0.73 0.07 4.2 5.0 4.0 5.0 0.2 0.1 <0.01 0.03 <0.01 0.93 0.69 0.60 0.56 0.21 0.44 0.48 6.3 185 29 9.5 5.1 132 254 540 43 121 187 107 95 59 67 158 107 39 29 272 8.0 165 27 7.8 5.4 139 221 484 50 162 260 151 102 91 49 152 152 47 34 379 1.3 11.7 1.5 0.5 0.6 7.2 18.7 44.3 6.7 8.3 13.8 11.3 5.5 5.5 2.8 12.5 15.7 4.0 3.1 15.2 <0.01 0.04 0.08 0.54 0.06 0.02 0.71 <0.01 0.22 0.65 0.05 0.56 0.35 0.19 <0.01 <0.01 0.57 0.41 0.87 <0.01 0.01 <0.01 0.94 0.48 0.88 0.06 0.97 0.16 0.97 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 0.38 0.08 0.48 0.24 0.86 0.84 0.17 <0.01 0.21 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 0.14 <0.01 <0.01 <0.01 <0.01 0.49 0.56 0.83 0.06 0.86 0.42 0.05 0.04 0.28 0.19 0.99 0.01 <0.01 0.14 0.78 <0.01 0.09 0.17 0.02 0.82 0.69 0.72 0.15 0.04 0.77 0.23 <0.01 0.16 0.79 0.64 0.88 0.96 0.03 0.52 0.56 0.32 0.38 0.21 0.04 0.48 Control NEAA + EAA EAA 6 6 6 0.68 176 0.88 198 2.9 5.1 10.8 157 28 7.5 5.4 142 205 533 41 75 122 75 61 38 48 117 71 29 19 163 ng/L 0.62 180 0.95 141 1.16 186 3.5 5.2 4.8 5.3 4.6 5.2 6.5 167 30 8.4 4.5 134 253 640 56 88 143 97 77 41 57 175 89 38 25 206 4.0 200 28 7.6 4.5 123 179 822 44 114 209 90 72 53 74 335 74 33 21 302 4.7 187 32 8.1 4.4 113 265 665 55 163 279 135 100 78 59 307 143 52 36 426 Plasma Urea Glucose Met × (Control vs. AA) EAA vs. NEAA + EAA mM M Met Ala Asn Asp α-Aminobutyric acid Glu Gln Gly His Ile Leu Lys Ornithine Phe Pro Ser Thr Trp Tyr Val 1 NEAA + EAA = 100 g/d nonessential + 100 g/d essential AA; EAA = 200 g/d essential AA. For n = 6. 2 other than Met, N balance responses to supplemental Met have been demonstrated to be linear up to 6 g/d (Campbell et al., 1997) and 10 g/d (Lambert et al., 2002). Also, the lack of the increase in retained N in response to the NEAA + EAA treatment when no supplemental Met was provided supports a conclusion that the responses to the AA treatments were not a result of supply of a colimiting AA. If deposited protein equals retained N × 6.25 and protein of tissue gain contains 2.0% Met (Ainslie et al., 1993), the calculated incremental efficiencies of use of the 4 g of supplemental Met/d were 16, 50, and 21% for steers receiving the control, NEAA + EAA, and EAA treatments, respectively. The 50% value for the NEAA + EAA treatment seems to be an anomaly resulting from the lack of response to this treatment in the absence of supplemental Met. Our efficiency of use of supplemental Met for the control animals (16%) was comparable to that previously reported in growing steers (Campbell et al., 1996; Awawdeh et al., 2004; Schroeder et al., 2006a) but was much less than the 66% predicted by the NRC (1996) equations. Effects of supplemental Met and AA on blood metabolites and hormones are presented in Table 4. Serum IGF-I concentration was not affected by treatments. Serum insulin and plasma urea concentrations increased (P < 0.01) in response to both AA treatments. Supplemental Met decreased (P ≤ 0.03) plasma concentrations of urea and glucose. Supplemental Met increased (P < 0.01) its plasma concentration as a result of increased supply, but the magnitude of increase was small enough to suggest that 4 g of Met/d did not exceed the steers’ requirement (Campbell et al., 1997; Lambert et al., 2002). Supplementation with Met decreased (P ≤ 0.05) plasma concentrations of Ala, Pro, Leu, and Val. Also, supplemental Met decreased (P < 0.01) plasma concentrations of Gly and Ser, but these decreases in response to Met were greater when steers received the AA treatments than when they received the control treatment [Met × (control vs. AA) interaction; P ≤ 0.04], likely because the greater concentrations achieved when AA treatments were infused allowed for greater responses to supplemental Met. Similar responses to supplemental Met 1806 Awawdeh et al. were observed for Leu, Ser, and Val in growing steers (Titgemeyer and Merchen, 1990; Campbell et al., 1997; Awawdeh et al., 2004). The decreases in Ala, Gly, Leu, Pro, Ser, and Val might be due to increased uptake and use of these AA for protein deposition as Met, the first limiting AA, became available in greater amounts. In part, the ability of supplemental Met to reduce plasma Ser concentrations also could be attributed to the use of Ser during of Met transsulfuration to produce cystathionine. However, the much greater concentrations of Ser in steers receiving no supplemental Met might also suggest that the elevated Ser concentrations are reflective of metabolic disturbances during Met deficiency and that, as expected, supplementation with the AA treatments exacerbated the Met deficiency. When Met supply was not limiting, excess AA did not substantially affect the plasma concentration of Ser (Lambert, 2001). Supplemental Met increased (P = 0.02) plasma concentrations of Glu. The lessened demands for N removal when supplemental Met was provided, as indicated by greater N retention, might have resulted in less amidation of Glu to Gln, although Gln concentrations were not consistently decreased in response to supplemental Met. Methionine tended (P = 0.06) to increase plasma αaminobutyric acid, likely produced from α-ketobutyric acid (Costa et al., 1985), which is a product of cystathionine cleavage. Plasma concentrations of Ala, Pro, and Ser increased (P < 0.01) in response to the NEAA + EAA treatment, likely as a result of increased supply. Also, some EAA (Ile, Leu, Phe, and Val) increased (P < 0.01) in response to the NEAA + EAA treatment, likely due to increased supply of these AA. Plasma concentration of ornithine increased in response to the NEAA + EAA treatment when supplemental Met was provided, but there was no effect when no supplemental Met was provided [Met × (control vs. AA) interaction; P < 0.01]. The EAA treatment increased plasma concentrations of Ile, Leu, Lys, Phe, Thr, Trp, Tyr, and Val above those observed with the NEAA + EAA treatment [(EAA vs. NEAA + EAA); P < 0.01], likely due to greater amounts infused. Also, plasma concentration of ornithine increased with the EAA treatment, and the increase was greater when supplemental Met was provided [Met × (control vs. AA) interaction; P < 0.01]. Although Ala and Ser were not included in the EAA treatment, the EAA treatment increased plasma concentration of these AA; however, the responses to the EAA treatment tended to be less (P ≤ 0.14) than that observed for the NEAA + EAA treatment. Most of the N resulting from catabolism of infused EAA should be removed from the system as urea. Because Ala acts as an interorgan transporter of N, the increase in plasma concentration of Ala in response to the EAA treatment might be a result of increased interorgan N transport. Both AA treatments decreased (P = 0.01) plasma concentrations of Met and tended (P = 0.06) to decrease Glu concentrations. The decreases in plasma Met concentrations in response to AA treatments could be ex- plained by an increased Met uptake and subsequent use in protein deposition, as indicated by increased retained N in response to these AA treatments. The decreases in plasma Glu concentrations in response to AA treatments might be a result of increased urea synthesis to remove excess N infused. The AA treatments provided AA beyond the AA requirements for protein deposition, as indicated by increased urinary urea (Table 3) and plasma urea concentrations (Table 4). Thus, excess AA must be removed from the system, mainly via ureagenesis. Glutamate N can be utilized in ureagenesis via transamination to aspartate (Parker et al., 1995), which will contribute 1 of the 2 N used in urea synthesis. The decrease in Glu concentration in response to AA treatments also might be due to increased amidation to Gln. Experiment 2 Diet digestibilities and N retention data in response to supplemental Leu and AA are presented in Table 5. Diet DM and OM digestibilities were not altered (P ≥ 0.13) by treatments and averaged 74.5 and 77.0%, respectively. Total N intake increased (P < 0.01) with AA treatments, as a result of additional N infused. Fecal N was not affected (P > 0.12) by treatments. There were no interactions (P ≥ 0.32) between supplemental Leu and AA treatments for urinary and retained N. Supplementation with 4 g of Leu/d increased (P < 0.01) retained N from 30.6 to 34.6 g/d, as a result of decreasing (P < 0.01) urinary N from 54.9 to 51.1 g/d. This was an expected response because Leu was purposefully designed to be the limiting AA (Löest et al., 2001; Awawdeh et al., 2004). Urinary urea N decreased (P < 0.01) from 45.3 to 40.1 g/d, but urinary NH3-N increased (P = 0.04) from 2.2 to 3.2 g/d in response to supplemental Leu. Although the increase in urinary NH3-N resulting from supplementation with Leu was significant, the low amounts of urinary NH3-N here, as well as in Exp. 1, indicate that our steers were not experiencing metabolic acidosis, which would have increased urinary NH3 excretion more dramatically (Vagnoni and Oetzel, 1998). Both AA treatments increased (P < 0.01) urinary excretion of N and urea N. However, the increases in urinary N in response to AA treatments (increase of 22.0 and 23.8 g/d) were less than the increases in intake N (26.4 g/d), resulting in increases (P = 0.01) in retained N in response to both AA treatments. Also, the increases in urinary N were greater (P = 0.05) in response to the EAA treatment than to the NEAA + EAA treatment, resulting in a greater (P = 0.13) retained N for the NEAA + EAA treatment than for the EAA treatment (increase of 4.3 vs. 2.2 g/d). Urinary NH3-N increased (P = 0.03) in response to both AA treatments. The increases in retained N in response to AA treatments might have resulted from supplying additional energy. Because retained N was increased at the same amounts of Leu, both AA treatments improved the efficiency of 1807 Amino acid utilization by cattle Table 5. Effect of supplemental Leu and AA on N balance and diet digestibility of growing steers (Exp. 2)1 P value No Leu Item n 4 g of Leu/d Control NEAA + EAA EAA Control NEAA + EAA EAA SEM2 Leu Control vs. AA EAA vs. EAA + NEAA Leu × (Control vs. AA) Leu × (EAA vs. EAA + NEAA) 7 5 6 5 6 6 Nitrogen, g/d Dietary Infused Total intake Fecal Urinary Urea Ammonia Retained 53.4 33.0 86.5 18.2 40.2 30.5 1.9 28.0 53.7 58.4 112.2 18.3 60.9 51.5 1.8 33.0 53.2 59.3 112.6 18.5 63.5 53.7 2.9 30.7 53.6 33.5 87.1 19.0 35.3 25.4 2.1 32.7 55.3 58.9 114.2 19.4 58.4 47.0 3.8 36.3 54.1 59.8 113.9 19.8 59.5 47.7 3.8 34.6 1.1 — 1.1 0.9 0.9 1.7 0.6 1.4 0.32 0.50 0.43 0.56 0.73 0.14 0.12 <0.01 <0.01 0.04 <0.01 <0.01 0.56 <0.01 <0.01 0.03 <0.01 0.93 0.69 0.05 0.28 0.32 0.13 0.56 0.75 0.32 0.95 0.25 0.66 0.73 0.95 0.42 0.58 0.38 0.81 Diet digestibility, % DM OM 74.1 76.6 74.8 77.5 74.7 77.2 75.2 77.7 75.1 77.5 73.1 75.6 1.0 1.1 0.96 0.76 0.70 0.70 0.17 0.13 0.25 0.17 0.22 0.27 1 NEAA + EAA = 100 g/d nonessential + 100 g/d essential AA; EAA = 200 g/d essential AA. For n = 6. 2 Leu use. Our observation that increased AA supply improved protein deposition when Leu limited N retention is in agreement with our findings in Exp. 1, in which Met was limiting, and with those of McCuistion et al. (2004), in which His was limiting. We expected that excess branched-chain AA (Val and Ile) might increase Leu catabolism by increasing branched-chain α-keto acid dehydrogenase (Block, 1989). In contrast, supplying a complete mixture of AA that contained Ile and Val in amounts greater than the steers’ requirements improved protein deposition in Exp. 2. Based on previous work from our laboratory (Awawdeh et al., 2005), 4 g of Leu/d was selected to be less than the animals’ requirements to ensure that steers were able to respond to supplemental Leu. If retained N was completely deposited as tissue protein (retained N × 6.25) and if tissue protein gain contains 6.7% Leu (Ainslie et al., 1993), the calculated incremental efficiencies of use of the 4 g of supplemental Leu/d averaged 41%, without large differences among the AA treatments (49, 34, and 41% for steers receiving the control, NEAA + EAA, and EAA treatments, respectively). The average value (41%) for the incremental efficiency of supplemental Leu use agreed with our previously reported values (Awawdeh et al., 2005) but was less than the 69% value predicted by the NRC (1996). The 41% value is greater than that for Met in Exp. 1 (16% for the control group), however, indicating that different AA might have different efficiency values. Effects of supplemental Leu and AA on blood metabolites and hormones are presented in Table 6. Serum IGF-I concentrations were not affected (P ≥ 0.19) by any treatment. Supplemental Leu decreased (P ≤ 0.03) plasma concentrations of urea and tended to increase (P = 0.06) serum insulin concentrations. Both AA treatments increased (P < 0.01) plasma urea concentrations, decreased (P < 0.01) plasma glucose concentrations, and tended (P = 0.10) to increase serum insulin concentrations. The insulin response to the AA treatments tended to be greater (P = 0.12) when 4 g of supplemental Leu/d was provided. Decreased glucose concentrations in response to AA treatments could reflect increased use of glucose in support of increased tissue deposition or responses to alterations in insulin. Plasma Leu concentrations increased (P < 0.01) in response to supplemental Leu, likely due to increased supply; the observed increase tended (P = 0.14) to be greater with AA treatments, predominantly due to lower concentrations achieved when AA treatments were infused, which allowed for greater responses to supplemental Leu. The magnitude of increase in plasma Leu in response to supplemental Leu was small enough to suggest that 4 g of Leu/d was less than the steers’ requirements. Supplementation with Leu decreased (P ≤ 0.02) plasma concentrations of Asn, Ile, Thr, Tyr, and Val, and tended (P ≤ 0.17) to decrease plasma Asp, Gly, Met, ornithine, and Ser concentrations, in large part resulting from increased uptake and use in protein deposition as Leu, the limiting AA, became more available. Plasma concentrations of Ile, Tyr, and Val in response to infused Leu were previously reported in growing steers (Greenwood and Titgemeyer, 2000; Löest et al., 2001; Awawdeh et al., 2005). The NEAA + EAA treatment increased (P < 0.01) plasma concentrations of Ala, Pro, and Ser, likely as a result of increased supply. Also, plasma concentrations of a number of EAA (Ile, Lys, Met, Phe, Thr, Trp, Tyr, and Val) increased (P ≤ 0.02) in response to the NEAA + EAA treatment, likely due to increased supply. The EAA treatment increased (P ≤ 0.02) plasma concentrations of Ile, Lys, Met, Phe, Thr, Trp, Tyr, and Val, compared with those of the NEAA + EAA treatment, likely due to greater amounts being infused. 1808 Awawdeh et al. Table 6. Effect of supplemental Leu and AA on serum hormone and plasma metabolite concentrations of growing steers (Exp. 2)1 P value No Leu Item 4 g of Leu/d Control NEAA + EAA EAA 7 5 6 n Serum Insulin IGF-I Leu × (Control vs. AA) Leu × (EAA vs. NEAA + EAA) SEM2 Leu Control vs. AA EAA vs. NEAA + EAA 0.99 260 0.1 54.2 0.06 0.67 0.10 0.44 0.87 0.19 0.12 0.43 0.91 0.30 3.6 5.44 0.3 0.1 0.03 0.35 <0.01 <0.01 0.07 0.76 0.90 0.32 0.14 0.20 3.8 24.9 2.1 0.72 1.1 6.8 17.7 39.0 4.0 13.2 12.9 3.2 5.2 4.4 3.1 11.0 9.5 2.6 2.1 26.6 <0.01 0.70 <0.01 0.09 0.29 0.91 0.27 0.08 0.44 <0.01 0.21 0.17 0.14 0.23 0.24 0.13 0.02 0.51 0.02 <0.01 0.05 0.04 0.89 <0.01 <0.01 0.92 0.32 0.29 0.05 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 0.28 <0.01 0.20 0.69 0.41 0.76 0.57 <0.01 0.83 <0.01 0.01 <0.01 0.02 <0.01 <0.01 <0.01 <0.01 0.02 <0.01 <0.01 0.14 0.81 0.40 0.41 0.56 0.79 0.35 0.10 0.05 0.21 0.54 0.27 0.40 0.94 0.22 0.87 0.91 0.16 0.45 0.32 0.39 0.99 0.42 0.10 0.74 0.64 0.51 0.85 0.18 0.14 0.20 0.91 0.41 0.73 0.88 0.12 0.51 0.30 0.22 0.36 Control NEAA + EAA EAA 5 6 6 0.65 262 0.99 269 4.1 5.32 ng/L 0.68 280 0.67 274 0.70 203 2.9 5.65 4.1 5.31 4.1 5.11 Plasma mM Urea Glucose 2.6 5.60 M Leu Ala Asn Asp α-Aminobutyric acid Glu Gln Gly His Ile Lys Met Ornithine Phe Pro Ser Thr Trp Tyr Val 31 213 33 7.4 11 85 258 450 42 130 85 25 54 41 52 65 69 41 20 273 23 267 34 10.3 14 85 241 511 40 253 114 32 77 62 68 105 113 47 25 529 22 213 31 8.9 14 90 223 349 44 300 124 40 84 78 47 80 137 51 30 672 37 203 28 7.1 9 87 227 414 33 103 71 25 45 37 46 54 55 35 15 207 38 263 29 7.9 13 86 227 478 46 194 99 28 71 56 67 106 104 45 20 422 33 211 29 8.8 14 85 228 307 41 266 129 36 84 75 46 50 119 55 30 592 1 NEAA + EAA = 100 g/d nonessential + 100 g/d essential AA; EAA = 200 g/d essential AA. For n = 6. 2 Both AA treatments decreased (P = 0.05) plasma concentrations of Leu, likely due to increased uptake and use of Leu in protein deposition, as indicated by increased N retention in response to AA treatments. Also, both AA treatments increased (P < 0.01) plasma concentrations of Asp and α-aminobutyric acid. The increases in plasma concentrations of α-aminobutyric acid in response to AA treatments might be a result of increased Met supply, inasmuch as α-aminobutyric acid production can result from Met transsulfuration. Plasma His concentrations were increased with AA treatments when supplemental Leu was provided but not without it [Leu × (control vs. AA) interaction; P = 0.05]. General Discussion We examined the effects of excess AA on the wholebody protein deposition under conditions in which either Met or Leu was limiting. To achieve that, the diet was formulated to provide deficient amounts of digestible AA, and then supplements containing all essential AA, except Met in Exp. 1 or Leu in Exp. 2, were pro- vided. If the AA under study were not limiting, negative effects of excess N, if any, on AA use might not lead to any changes in performance because an excess supply of the AA under study could allow for optimal performance, even in the face of reduced efficiency of use. For example, Hagemeier et al. (1983) demonstrated that supply of excess Arg had no negative effect on swine performance when Lys supply was adequate, but when Lys supply was limiting, excess Arg decreased performance. Similarly, excess Leu decreased retained N in pigs when Ile supply was limiting, but did not affect retained N when Ile was not limiting (Langer and Fuller, 2000). We expected that excess AA might decrease retained N as a result of increased catabolism of the limiting AA to support ureagenesis or as a result of an AA imbalance or both. Various mixtures of excess AA have led to AA imbalances and, subsequently, to decreased performance of chicks (Park and Austic, 2000), rats and mice (Sauberlich, 1956; Peng, 1979), and pigs (Southern and Baker, 1982; Hagemeier et al., 1983; Edmonds and Baker, 1987). Supplying excess Met (0.177 g of Met/kg Amino acid utilization by cattle of BW) decreased retained N in Holstein calves (Abe et al., 1999). In Exp. 2, the greatest amount of Met (EAA treatment) was roughly two-thirds of that provided by Abe et al. (1999; 0.12 vs. 0.177 g/kg of BW). Also, supplying excess Lys (0.43 g/kg of BW) did not decrease retained N in cattle (Abe et al., 2001). In our experiments, supplemental Lys was provided at 0.23 g/kg of BW in Exp. 1 and 0.29 g/kg of BW in Exp. 2 for the EAA treatment. The discrepancy between our findings and those of studies that observed negative effects of supplying excess AA (leading to AA imbalance) can possibly be attributed to species differences, to the supply of single AA vs. mixtures containing all EAA (except the limiting AA under investigation), or to the amount of excess AA provided. In our study, excess AA improved protein deposition when Leu supply limited steers’ performance (Exp. 2). When Met supply limited steers’ performance (Exp. 1), EAA increased protein deposition, regardless of supplemental Met, but excess NEAA + EAA increased protein deposition only when 4 g of supplemental Met/d was provided. When His supply was very limited (no additional His was provided), only a mixture of NEAA and EAA, but not EAA alone, increased protein deposition (McCuistion et al., 2004). It is clear that the effects on protein deposition of supplying excess AA are dependent upon the AA being studied and on the source of excess AA. We demonstrated that excess AA improved the efficiency of use of Met and Leu because retained N increased at the same amounts of the limiting AA. The efficiency of use of Met from the basal diet in Exp. 1 was 105% for the control and increased to an average of 121% for the NEAA + EAA and EAA treatments. The basal Met supply was 2.7 g/d provided by the diet (Campbell et al., 1997), and the amounts of Met deposited in the body were calculated assuming that deposited protein equaled retained N × 6.25 and that protein of tissue gain contained 2.0% Met (Ainslie et al., 1993). Similarly, additional AA included in the AA treatments in Exp. 2 increased the efficiency of use of the basal dietary Leu from 81% for control to an average of 92% for the NEAA + EAA and EAA treatments. The basal Leu supply was 14.4 g/d provided by the diet (Campbell et al., 1997), and the amounts of Leu deposited in the body were calculated assuming that deposited protein equaled retained N × 6.25 and that protein of tissue gain contained 6.7% Leu (Ainslie et al., 1993). For both Met and Leu, the efficiencies for use of the basal AA supply are likely overestimated due to the routine overestimation of protein deposition by the N balance technique. The improvements in efficiency in response to the NEAA + EAA and EAA treatments could be explained as a result of excess AA being used as a source of energy. Gerrits et al. (1996) demonstrated that protein deposition was improved in preruminant calves by supplying a protein-free energy source when the protein supply was limiting, indicating that supplemental energy improves the use efficiency of dietary protein for 1809 tissue deposition. Increasing the energy supply linearly increased retained N in growing steers limited by Met, resulting in improved use efficiency of Met (Schroeder et al., 2006a). The efficiency of use of the limiting AA may be improved because energy supply determines the protein turnover rate in ruminants by decreasing protein degradation and, subsequently, increasing protein deposition (Asplund, 1994). In our study, additional AA N included in the AA treatments was used for protein deposition with poor efficiencies of 21% (Exp. 1) and 11% (Exp. 2), suggesting that they were not used predominantly as a source of limiting AA. When all supplemental energy provided to pigs was from protein, the efficiency of protein use was 17% (Fuller and Crofts, 1977), similar to our values. It is also possible that the net effects observed in response to the AA treatments were a balance of benefits resulting from the energy supplied and of detriments from AA imbalances or increases in AA catabolism. If both positive and negative effects were present, it is clear that the positive effects outweighed the negative ones. Because we did not provide energy to the control treatment in amounts equal to the AA treatments, it is impossible to separate these responses. The effects of supplemental Met, Leu, and AA on plasma glucose and serum insulin levels are dependent on the AA being studied and on the protein (AA) status of the animals. For example, Met decreased plasma glucose but had no effect on serum insulin levels (Exp. 1). On the other hand, Leu increased serum insulin but did not affect plasma glucose levels (Exp. 2). Although the same AA mixtures, except for the limiting AA, were used in both experiments, excess AA had different effects on glucose and insulin. Excess AA increased serum insulin in both experiments, but decreased plasma glucose only when Leu was limiting (Exp. 2). Different AA have different use efficiency values, and the efficiency values, at least for Leu and Met, are less than those predicted by the NRC (1996). Our estimated efficiencies of use for Leu and Met raise doubts about the validity of using the single equation for all AA developed by Ainslie et al. (1993) and adopted by the NRC (1996). We previously studied the effects of ruminal NH3 loading on protein deposition in growing steers limited by Met, Leu, or His supply (Awawdeh et al., 2004, 2005; McCuistion et al., 2004). From the current study and that of McCuistion et al. (2004), excess N from AA does not negatively affect protein deposition by cattle, at least when His, Leu, or Met is limiting. This finding contrasts the suggestions of in vitro (Mutsvangwa et al., 1996, 1999) and in vivo (Lobley et al., 1995) studies that excess N might lead to inefficient use of dietary AA as a result of metabolically consuming AA to provide α-amino N to support ureagenesis. 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