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. 2019 Jul 3;97(9):3611–3616. doi: 10.1093/jas/skz227

Ruminal microbes of adult steers do not degrade extracellular L-citrulline and have a limited ability to metabolize extracellular L-glutamate1,2

Kyler R Gilbreath 1, Gayan I Nawaratna 1, Tryon A Wickersham 1, M Carey Satterfield 1, Fuller W Bazer 1, Guoyao Wu 1,✉
PMCID: PMC6735895  PMID: 31269197

Abstract

The microbial population within the rumen has long been considered to have the capability of extensively degrading all dietary AA. Results from our feeding trials revealed that this dogma is not correct. In vitro studies were conducted to test the hypothesis that certain AA undergo little degradation by ruminal microbes. Whole ruminal fluid (3 mL, containing microorganisms) from cannulated adult steers (~500 kg, n = 6) was incubated at 37 °C with 5 mM l-glutamine, l-glutamate, l-arginine, or l-citrulline for 0, 0.5, 1, and 2 h to determine time-dependent changes in the metabolism of these AA. Additional ruminal fluid was incubated with 0, 0.5, 2 or 5 mM l-glutamine, l-glutamate, l-arginine, or l-citrulline for 2 h to determine dose-dependent changes in their metabolism. An aliquot (50 µL) of the incubation solution was collected at the predetermined time points for AA analyses. There was extensive hydrolysis of l-glutamine into l-glutamate and ammonia, and l-arginine into l-ornithine, l-proline, and ammonia, but the near absence of catabolism of extracellular l-glutamate and no degradation of extracellular l-citrulline by ruminal microbes. There was little uptake of 14C-labeled l-glutamate and no detectable uptake of 14C-labeled l-citrulline by the cells. These results indicate, for the first time, that ruminal microbes of adult steers do not degrade extracellular l-citrulline and that metabolism of extracellular l-glutamate is negligible compared with their ability to extensively catabolize extracellular l-arginine and l-glutamine.

Keywords: amino acids, arginine, degradation, ruminal bacteria

INTRODUCTION

In ruminants, dietary protein is hydrolyzed by bacterial proteases and peptidases into small peptides and AA in the rumen, whereas free AA are further degraded to ammonia and their carbon skeletons by a number of microbial enzymes, including deaminases, transaminases, hydrolases, and decarboxylases (Owens and Bergen, 1983; Wu, 2013). In the presence of α-ketoacids (e.g., pyruvate, oxaloacetate, and α-ketoglutarate which are products of carbohydrate metabolism) and sulfur, ammonia is utilized by ruminal bacteria for the synthesis of new AA and proteins (Wu, 2018). The rates of AA catabolism are high in the rumen, such that all AA studied to date (Ala, Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Orn, Phe, Pro, Ser, Thr, Tyr, and Val) do not escape the rumen (Lewis, 1955; Annison, 1956; Chalupa, 1975, 1976; Butter and Folds, 1985; Cozzi et al., 1995; Bach et al., 2005). Thus, the microbial population within the rumen has long been considered to have the capability of extensively degrading all dietary AA (Lewis and Emery, 1962; Chalupa, 1976; Scheifinger et al., 1976; Schwab et al., 1976; Kung and Rode, 1996; NRC, 2000, 2001). However, we are not aware of studies regarding the degradability of extracellular l-citrulline (Cit) or l-glutamate (Glu) by ruminal microorganisms (primarily bacteria). Results of our feeding trials indicated that extracellular Cit and Glu may not be metabolized in the rumen of adult steers (Gilbreath et al., 2017) or beef cows (Wu et al., 2018). Therefore, the objective of the present study was to determine whether Cit and Glu are catabolized by ruminal microbes.

MATERIALS AND METHODS

This study was approved by the Institutional Animal Care and Use Committee of Texas A&M University. The research protocol followed the United States Department of Agriculture animal welfare regulations.

Angus steers (~500 kg) had free access to hay and drinking water (Bell et al., 2017). Ruminal fluid (containing microorganisms) was obtained from 6 cannulated steers (n = 6) that had been deprived of food for 16 h. Specifically, whole ruminal samples were collected from steers with a suction strainer (19 mm diameter; 1.5 mm mesh for its filter), as described by Bell et al. (2017). Ruminal samples from each steer were gently mixed and then rapidly transferred into 50-mL polypropylene tubes. This ruminal fluid (containing microorganisms) was then aliquoted into polypropylene tubes (3 mL per tube), which contained one of the following: no addition of AA, 2.2 mg l-glutamine (5 mM), 2.2 mg Glu (5 mM), 3.1 mg l-arginine-HCl (5 mM), or 2.7 mg Cit (5 mM). No carbohydrate or sulfur compounds were added to the incubation medium. The tubes were gassed with CO2 for 20 s, capped, and then incubated (70 oscillations/min) at 37 °C. At 0, 0.5, 1, and 2 h of the incubation, an aliquot (50 µL) of the incubation solution was collected from the tube without opening the cap.

To determine the concentration-dependent changes in AA catabolism, a 5 mM AA solution was diluted with ruminal fluid to yield 0.5 and 2 mM solutions. No carbohydrate or sulfur compounds were added to the incubation medium. The solutions (in polypropylene tubes) were gassed with CO2 for 20 s, capped, and then incubated (70 oscillations/min) at 37 °C. At the end of a 2-h incubation period, an aliquot (50 µL) of the incubation solution was collected from the tube.

For AA analysis, an aliquot (20 µL) of 1.5 M HClO4 was added to 20 µL of the collected incubation sample, followed by the addition of 10 µL of 2 M K2CO3 for neutralization and then 150 µL of HPLC-grade water. The extracts were stored at −20 °C until analyzed for AA by HPLC (Wu and Meininger, 2008) and for urea using urease and Glu dehydrogenase (Wu, 1995).

To determine the uptake of AA by ruminal microbes, ruminal fluid (1 mL; containing microorganisms) was incubated as described earlier, except that the medium contained 5 mM l-[U-14C]Glu, l-[U-14C]Cit, l-[U-14C]arginine, or l-[U-14C]glutamine (100 dpm/nmol) for 2 h. The tracers were purchased from American Radiolabeled Chemicals Inc. (St. Louis, MO) and purified before use (Chen et al., 2009). Blanks, in which Krebs-bicarbonate buffer replaced ruminal fluid, were included for parallel incubation. At the end of the incubation period, 0.2 mL of 1.5 M HClO4 was added through the stopper into the incubation medium and 14CO2 was collected into Soluene, as we described previously (Li et al., 2016). Thereafter, the acidified medium was neutralized with 0.1 mL of 2 M K2CO3 as described earlier. An aliquot (100 µL of the whole extract) was analyzed for separation and collection of AA fractions using HPLC (Wu et al., 2000), as well as for 14C-urea using urease and Glu dehydrogenase (Wu and Brosnan, 1992). Incorporation of 14C-AA into microbial protein was determined by measuring the radioactivity of 14C-labeled proteins, as we described previously (Dai et al., 2010). The sum of labeled products was used to calculate the uptake of each radiolabeled substrate.

Data were analyzed by 1-way analysis of variance for repeated-measures data (Lee et al., 2019). Log transformation of variables was performed when the variance of data was not homogenous using the Levene’s test (Wei et al., 2012). Differences among treatment means were determined by the Student–Newman–Keuls multiple comparison test. P-values of ≤0.05 indicated statistical significance.

RESULTS AND DISCUSSION

l-Glutamine and l-arginine were extensively degraded by ruminal microbes in a time- and concentration-dependent manner (Tables 1 and 2). The major products of glutamine catabolism were Glu, ammonia, and alanine, whereas the major products of arginine catabolism were ornithine, Pro, and ammonia (Tables 1–3). At the end of the 2-h incubation, no urea or 14C-urea was detected in the medium. These results indicated that microorganisms (containing high urease activity to hydrolyze urea into ammonia and CO2; Firkins et al., 2007) present in the ruminal fluid were biochemically viable. In the current work, we did not measure the production of short-chain fatty acids by ruminal microbes.

Table 1.

Amino acid profile in the incubated steer ruminal fluid after addition of an AA1

Addition to medium Hour Asp Glu Gln Cit Arg Ornithine Ammonia2
None 0 14.3 ± 1.4d 46.0 ± 2.5 32.5 ± 3.1a 5.70 ± 0.71 18.2 ± 2.0 19.2 ± 2.4 0.72 ± 0.08
0.5 19.1 ± 1.6c 47.2 ± 3.7 30.7 ± 2.0ab 6.38 ± 0.84 16.8 ± 2.2 22.3 ± 3.4 0.73 ± 0.09
1 23.5 ± 1.7b 51.6 ± 5.0 28.3 ± 3.7bc 6.26 ± 0.64 17.2 ± 1.9 22.9 ± 3.7 0.75 ± 0.11
2 27.0 ± 1.9a 53.8 ± 4.5 25.3 ± 3.7c 6.51 ± 0.72 16.7 ± 2.1 24.2 ± 3.9 0.71 ± 0.08
P-value <0.001 0.409 <0.001 0.218 0.336 0.166 0.920
5 mM Gln 0 14.1 ± 1.3d 46.5 ± 2.7d 5,023 ± 9.0a 5.62 ± 0.73c 18.6 ± 2.2 19.4 ± 2.3c 0.71 ± 0.07d
0.5 20.9 ± 2.8c 726 ± 34c 4,218 ± 11b 6.14 ± 0.88bc 17.5 ± 2.3 25.1 ± 2.5b 1.37 ± 0.08c
1 27.4 ± 2.9b 1,082 ± 41b 3,950 ± 36c 6.82 ± 1.0ab 19.8 ± 1.9 27.3 ± 2.5ab 1.60 ± 0.06b
2 33.6 ± 3.2a 1,439 ± 47a 3,447 ± 44d 7.34 ± 0.82a 20.2 ± 1.8 29.1 ± 2.7a 1.82 ± 0.06a
P-value <0.001 <0.001 <0.001 0.036 0.527 <0.001 <0.001
5 mM Cit 0 14.6 ± 1.5d 46.3 ± 2.6 32.8 ± 3.2a 5,007 ± 7.0 18.4 ± 2.2 20.3 ± 2.6 0.73 ± 0.09
0.5 20.6 ± 2.3c 52.6 ± 3.4 32.5 ± 3.3a 4,996 ± 12 17.3 ± 2.1 24.3 ± 2.8 0.71 ± 0.08
1 26.2 ± 3.1b 53.2 ± 3.3 29.4 ± 2.7ab 5,003 ± 15 18.6 ± 2.1 23.5 ± 3.5 0.74 ± 0.10
2 31.8 ± 3.6a 55.1 ± 3.0 26.2 ± 2.4b 4,992 ± 13 19.3 ± 2.2 24.7 ± 3.7 0.75 ± 0.06
P-value <0.001 0.384 0.015 0.857 0.880 0.355 0.892
5 mM Glu 0 14.5 ± 1.6d 5,052 ± 31 32.2 ± 2.9a 5.81 ± 0.86 18.2 ± 2.1 19.6 ± 2.5 0.72 ± 0.08
0.5 19.8 ± 2.8c 5,029 ± 16 31.7 ± 3.0a 6.22 ± 0.93 18.0 ± 1.9 20.3 ± 2.7 0.72 ± 0.07
1 26.2 ± 2.4b 5,036 ± 10 28.4 ± 2.6ab 7.35 ± 0.90 18.4 ± 2.2 22.6 ± 3.8 0.73 ± 0.11
2 31.5 ± 2.7a 5,047 ± 28 25.4 ± 2.3b 7.27 ± 0.98 18.6 ± 2.6 24.3 ± 2.6 0.74 ± 0.09
P-value <0.001 0.973 0.018 0.493 0.972 0.346 0.910
5 mM Arg 0 14.7 ± 1.3d 45.2 ± 2.6 32.0 ± 2.8a 5.79 ± 0.77d 5,016 ± 9.0a 19.8 ± 2.2d 0.73 ± 0.09d
0.5 21.2 ± 2.6c 47.5 ± 1.3 31.4 ± 2.6a 27.8 ± 2.9c 4,636 ± 29b 44.7 ± 1.2c 1.36 ± 0.14c
1 27.5 ± 3.0b 46.6 ± 1.1 28.6 ± 2.4ab 41.0 ± 3.3b 4,467 ± 26c 58.8 ± 2.6b 1.57 ± 0.12b
2 32.7 ± 3.4a 47.8 ± 1.0 23.3 ± 2.2c 54.0 ± 4.2a 4,253 ± 23d 73.0 ± 4.0a 1.89 ± 0.10a
P-value <0.001 0.926 0.008 <0.001 <0.001 <0.001 <0.001
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1Ruminal fluid (containing microorganisms) was incubated for 0, 0.5, 1, and 2 h in the presence of an added AA (5 mM). Values, expressed as nmol/mL of ruminal fluid for AA and µmol/mL of ruminal fluid for ammonia, are means ± SEM, n = 6.

2NH3 plus NH4+.

a–dWithin a column, means not sharing the same superscript differ (P < 0.05).

Table 2.

Amino acid profile in the incubated steer ruminal fluid after addition of various concentrations of an AA1

Addition to medium Concentration, mM Asp Glu Gln Cit Arg Ornithine Ammonia2
Gln 0 27.0 ± 1.9b 53.8 ± 4.5d 25.3 ± 3.7d 6.51 ± 0.72b 16.7 ± 2.1b 24.2 ± 3.9b 0.71 ± 0.08d
0.5 29.3 ± 2.1ab 261 ± 12c 317 ± 18c 6.90 ± 0.75ab 18.0 ± 2.3ab 25.1 ± 2.5b 0.94 ± 0.07c
2 31.5 ± 2.6ab 693 ± 26b 1,402 ± 31b 7.13 ± 0.90ab 19.2 ± 2.5ab 27.3 ± 2.5ab 1.42 ± 0.07b
5 33.6 ± 3.2a 1,439 ± 47a 3,447 ± 44a 7.34 ± 0.82a 20.2 ± 1.8a 29.1 ± 2.7a 1.82 ± 0.06a
P-value <0.001 <0.001 <0.001 0.036 0.024 0.031 <0.001
Cit 0 27.0 ± 1.9 53.8 ± 4.5 25.3 ± 3.7 6.51 ± 0.72d 16.7 ± 2.1 24.2 ± 3.9 0.71 ± 0.08
0.5 29.4 ± 3.5 53.2 ± 4.2 26.1 ± 3.6 503 ± 8c 17.0 ± 2.8 22.9 ± 3.5 0.73 ± 0.09
2 28.8 ± 3.8 54.7 ± 4.7 25.9 ± 3.2 2,008 ± 11b 17.9 ± 3.0 23.1 ± 3.8 0.72 ± 0.11
5 31.8 ± 3.6 55.1 ± 3.0 26.2 ± 2.4 4,992 ± 13a 19.3 ± 2.2 24.7 ± 3.7 0.75 ± 0.06
P-value 0.246 0.887 0.924 <0.001 0.218 0.955 0.892
Glu 0 27.0 ± 1.9b 53.8 ± 4.5d 25.3 ± 3.7 6.51 ± 0.72 16.7 ± 2.1 24.2 ± 3.9 0.71 ± 0.08
0.5 29.4 ± 2.0ab 546 ± 12c 26.0 ± 3.3 6.88 ± 1.0 17.4 ± 2.5 23.9 ± 3.4 0.72 ± 0.09
2 30.7 ± 2.4ab 2,051 ± 14b 26.3 ± 3.0 6.75 ± 0.94 17.2 ± 2.3 25.1 ± 3.2 0.76 ± 0.10
5 31.5 ± 2.7a 5,047 ± 28a 25.4 ± 2.3 7.27 ± 0.98 18.6 ± 2.6 24.3 ± 2.6 0.74 ± 0.09
P-value <0.001 <0.001 0.986 0.905 0.921 0.824 0.915
Arg 0 27.0 ± 1.9b 53.8 ± 4.5 25.3 ± 3.7 6.51 ± 0.72d 16.7 ± 2.1d 24.2 ± 3.9d 0.71 ± 0.08d
0.5 28.4 ± 2.0b 49.2 ± 4.6 24.6 ± 3.9 27.8 ± 2.9c 401 ± 15c 44.7 ± 1.2c 0.93 ± 0.09c
2 30.9 ± 2.2ab 48.5 ± 4.2 25.0 ± 3.2 41.0 ± 3.3b 1,795 ± 20b 58.8 ± 2.6b 1.15 ± 0.10b
5 32.7 ± 3.4a 47.8 ± 1.0 23.3 ± 2.2 54.0 ± 4.2a 4,253 ± 23a 73.0 ± 4.0a 1.89 ± 0.10a
P-value <0.001 0.326 0.841 <0.001 <0.001 <0.001 <0.001
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1Ruminal fluid (containing microorganisms) was incubated for 2 h in the presence of an added AA (0, 0.5, 2, and 5 mM). Values, expressed as nmol/mL of ruminal fluid for AA and µmol/mL of ruminal fluid for ammonia, are means ± SEM, n = 6.

2NH3 plus NH4+.

a–dWithin a column, means not sharing the same superscript differ (P < 0.05).

Table 3.

Metabolism of 14C-labeled glutamate, citrulline, arginine, and glutamine in the incubated steer ruminal fluid1

Variable l-[U-14C]Glutamate (5 mM) l-[U-14C]Citrulline (5 mM) l-[U-14C]Arginine (5 mM) l-[U-14C]Glutamine (5 mM)
Uptake of AA 11.2 ± 1.0c ND2 780 ± 63b 1,583 ± 96a
Production of metabolites
  14CO2 1.62 ± 0.14c ND 108 ± 15b 191 ± 19a
  14C-Gln 0.64 ± 0.07 ND 1.03 ± 0.14 —
  14C-Asp 1.97 ± 0.15c ND 16.6 ± 1.2b 22.9 ± 2.5a
  14C-Ala 2.35 ± 0.37b ND 2.20 ± 0.34b 17.9 ± 2.1a
  14C-Glu — ND 2.73 ± 0.30b 1,395 ± 72a
  14C-Pro 0.16 ± 0.012c ND 572 ± 49a 2.40 ± 0.26b
  14C-Ornithine 0.22 ± 0.018c ND 48.8 ± 7.2a 12.1 ± 1.5b
  14C-Citrulline ND ND 47.3 ± 7.9a 1.77 ± 0.20b
  14C-Arg ND ND — 2.63 ± 0.32a
  14C-Protein3 6.85 ± 0.73c ND 78.4 ± 3.5b 102.2 ± 8.4a
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1Ruminal fluid (containing microorganisms) was incubated for 2 h in the presence of a 14C-labeled AA plus an unlabeled AA (5 mM). Values, expressed as nmol/mL of ruminal fluid per 2 h, are means ± SEM, n = 6.

2ND = not detected.

3Incorporation of the labeled AA into protein.

a-cWithin a row, means not sharing the same superscript are different (P < 0.05).

Concentrations of Pro in the medium containing 5 mM arginine were 19.3 ± 1.7, 316 ± 25, 488 ± 36, and 632 ± 59 nmol/mL of ruminal fluid (mean ± SEM, n = 6), respectively, after the 0, 0.5, 1, and 2 h of incubation (P < 0.01). At the end of the 2-h incubation, concentrations of Pro in the medium containing 0.5 and 2 mM arginine were 85 ± 8.0 and 279 ± 31 nmol/mL of ruminal fluid, respectively. In contrast, there was no detectable loss of extracellular Cit or Glu from the ruminal fluid during a 2-h period of incubation (Tables 1 and 2). Consistently, the rates of formation of 14CO2, 14C-Gln, 14C-Asp, 14C-Ala, 14C-ornithine, 14C-Pro, and 14C-protein from extracellular 14C-Glu (5 mM) were negligible and there was no detectable production of 14C-Cit or 14C-arginine from extracellular 14C-Glu, compared with the formation of 14C-labeled products from extracellular 14C-glutamine or 14C-arginine (Table 3).

There was no formation of 14C-labeled AA (including Asn, Ser, Gly, His, Cit, Arg, Thr, taurine, Phe, Leu, Ile, Val, Trp, Tyr, Met, or Cys) from 5 mM 14C-Glu during a 2-h period of incubation. There was no detectable formation of 14CO2 or any 14C-labeled AA (including Glu, Asp, Asn, Ser, Gln, His, Gly, Arg, Thr, taurine, Ala, Phe, Leu, Ile, Val, Trp, Tyr, ornithine, Lys, Pro, Met, or Cys) from extracellular 14C-Cit (5 mM) during a 2-h period of incubation. Collectively, these results indicated that extracellular Glu and Cit underwent little or no degradation by microbes in the rumen of steers despite their high ability to degrade extracellular 14C-glutamine (5 mM) and 14C-arginine (5 mM). We are not aware of work on the recovery of Cit in the abomasum after its feeding to ruminants.

The sum of the Glu products (11.2 nmol Glu/mL of ruminal fluid) represented only 0.22% of Glu present in the incubation medium (5,000 nmol Glu/mL of ruminal fluid) and 0.72% of the rate of Gln hydrolysis into Glu (1,553 mol Gln/mL of ruminal fluid per 2 h; mean ± SEM, n = 6). The ruminal bacterium Streptococcus bovis may contribute to a slow rate of utilization of extracellular Glu (Hen and Russell, 1989). Although the ruminal microbes of steers metabolized little or no extracellular Glu, glutamine-derived intracellular Glu can be degraded to CO2, aspartate, and ornithine (Table 2). However, most of the glutamine-derived Glu accumulated in the bovine ruminal fluid, possibly due to a limited availability of intracellular α-ketoacids for initiating Glu transamination in the microbes. Likewise, our results indicate that bovine ruminal microbes do not take up extracellular Cit possibly due to the lack of a transporter. In support of this view, Stalon and Mercenier (1984) reported that few bacteria can utilize extracellular Cit as a nitrogen or carbon source. Thus, in ruminants, orally administered Cit and Glu may effectively enter the small intestine to exert their functions on the mucosa, as demonstrated in nonruminants (Wu, 2013). Thus, there is no need to encapsulate Cit or Glu in a rumen-protected form when they are supplemented to the diet of ruminants. Dietary Cit may be an effective precursor of arginine in ruminants to elicit physiological responses such as improvements in embryonic survival, immunity, and lactation, as reported for swine (Wu et al., 2018). With such a simple means to provide ruminants with dietary Cit and Glu, there is no need to manufacture rumen-protected arginine through the use of challenging and expensive technologies. Sources of dietary Cit may be crystalline Cit (Wu, 2013) and watermelon by-products (Wu et al., 2007). Likewise, Glu can be supplemented to the diets of ruminants to improve intestinal function, without the need of its intragastric administration (Tagari and Bergman, 1978) or duodenal infusion (Brake et al., 2014).

To our knowledge, this is the first report on the degradability of extracellular Cit and Glu by mixed microbes in the rumen of any species. However, Chalupa (1976) demonstrated that bovine ruminal fluid (containing microorganisms), when incubated in the presence of 2% starch plus 8 mM urea, utilized extracellular Glu based on the disappearance of Glu from the medium over a 6-h period. Those results were cautiously interpreted as the “apparent degradation” of Glu by microbes in the rumen, due to the lack of evidence for true degradation of this AA such as the formation of metabolites from Glu. Chalupa (1976) provided urea (a ready precursor of ammonia) and starch (a ready source of carbon skeletons including α-ketoacids) to support synthetic processes; therefore, the disappearance of Glu from the incubation medium may have resulted primarily from the utilization of extracellular Glu for protein or peptide synthesis by microbes in the rumen, rather than degradation of extracellular Glu.

In summary, the results of our experiments indicated that, in contrast to glutamine and arginine, extracellular Cit and Glu do not undergo significant catabolism by microorganisms (primarily bacteria) in the rumen of steers. These findings correct the traditional view that all AA in diets are extensively degraded in the rumen. This new concept has far-reaching implications in the nutrition of ruminants and their dietary supplementation with selected AA to enhance production efficiencies such as growth, pregnancy, and lactation. Thus, Cit, without encapsulation or protection from ruminal microbes, may be effectively supplemented to the diets of ruminants to increase the concentrations of Cit and arginine in plasma. Dietary Cit (a stable and neutral nonproteinogenic AA) may be an excellent source of arginine for utilization and metabolism by ruminants to enhance their productivity. Similarly, Glu may be added to the diet of ruminants for enhancing their growth and production performance.

Footnotes

1

Supported by the Texas A&M AgriLife Research Beef Program and the Animal Science Mini-Grant Program.

2

We thank W. David Long, Neil D. Wu, and Shengdi Hu for technical assistance.

LITERATURE CITED

  1. Annison E. F. 1956. Nitrogen metabolism in the sheep; protein digestion in the rumen. Biochem. J. 64:705–714. doi: 10.1042/bj0640705 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bach A., Calsamiglia S., and Stern M. D.. 2005. Nitrogen metabolism in the rumen. J. Dairy Sci. 88(Suppl 1):E9–E21. doi: 10.3168/jds.S0022-0302(05)73133-7 [DOI] [PubMed] [Google Scholar]
  3. Bell N. L., Callaway T. R., Anderson R. C., Franco M. O., Sawyer J. E., and Wickersham T. A.. 2017. Effect of monensin withdrawal on intake, digestion, and ruminal fermentation parameters by Bos taurus indicus and Bos taurus taurus steers consuming bermudagrass hay. J. Anim. Sci. 95:2747–2757. doi: 10.2527/jas.2016.1013 [DOI] [PubMed] [Google Scholar]
  4. Brake D. W., Titgemeyer E. C., and Anderson D. E.. 2014. Duodenal supply of glutamate and casein both improve intestinal starch digestion in cattle but by apparently different mechanisms. J. Anim. Sci. 92:4057–4067. doi: 10.2527/jas.2014-7909 [DOI] [PubMed] [Google Scholar]
  5. Butter P. J., and Folds A. N.. 1985. Amino acid requirements of ruminants. In: Haresign W. and Cole D. J. A. editors, Recent advances in animal nutrition. Butterworth-Heinemann Ltd., Oxford, UK. p. 257–271. [Google Scholar]
  6. Chalupa W. 1975. Rumen bypass and protection of proteins and amino acids. J. Dairy Sci. 58:1198–1218. doi: 10.3168/jds.S0022-0302(75)84697-2 [DOI] [PubMed] [Google Scholar]
  7. Chalupa W. 1976. Degradation of amino acids by the mixed rumen microbial population. J. Anim. Sci. 43:828–834. doi: 10.2527/jas1976.434828x [DOI] [PubMed] [Google Scholar]
  8. Chen L., Li P., Wang J., Li X., Gao H., Yin Y., Hou Y., and Wu G.. 2009. Catabolism of nutritionally essential amino acids in developing porcine enterocytes. Amino Acids 37:143–152. doi: 10.1007/s00726-009-0268-1 [DOI] [PubMed] [Google Scholar]
  9. Cozzi G., Andrighetto I., Berzaghi P., and Polan C. E.. 1995. In situ ruminal disappearance of essential amino acids in protein feedstuffs. J. Dairy Sci. 78:161–171. doi: 10.3168/jds.S0022-0302(95)76626-7 [DOI] [PubMed] [Google Scholar]
  10. Dai Z. L., Zhang J., Wu G., and Zhu W. Y.. 2010. Utilization of amino acids by bacteria from the pig small intestine. Amino Acids 39:1201–1215. doi: 10.1007/s00726-010-0556-9 [DOI] [PubMed] [Google Scholar]
  11. Firkins J. L., Yu Z., and Morrison M.. 2007. Ruminal nitrogen metabolism: Perspectives for integration of microbiology and nutrition for dairy. J. Dairy Sci. 90(Suppl. 1):E1–E16. doi: 10.3168/jds.2006-518 [DOI] [PubMed] [Google Scholar]
  12. Gilbreath K. R., Nawaratna G., Wickersham T. A., Satterfield M. C., Bazer F. W., and Wu G.. 2017. Ruminal microbes of adult steers extensively degrade L-glutamine but not L-glutamate or L-citrulline. J. Anim. Sci. 95(Suppl. 4):35. doi: 10.2527/asasann.2017.070 [DOI] [Google Scholar]
  13. Hen G., and Russell J. B.. 1989. Transport and metabolism of glutamine and glutamate by the rumen bacterium, Streptococcus bovis. J. Bacteriol. 171:2981–2985. doi:10.1128/jb.171.6.2981-2985.1989 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kung L., and Rode L. M.. 1996. Amino acid metabolism in ruminants. Anim. Feed Sci. Tech. 59:167–172. [Google Scholar]
  15. Lee U., Garcia T. P., Carroll R. J., Gilbreath K. R., and Wu G.. 2019. Analysis of repeated measures data in nutrition research. Front. Biosci. 24:1377–1389. doi:10.2741/4785 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lewis D. 1955. Amino-acid metabolism in the rumen of the sheep. Br. J. Nutr. 9:215–230. [DOI] [PubMed] [Google Scholar]
  17. Lewis T. R., and Emery R. S.. 1962. Metabolism of amino acids in the bovine rumen. J. Dairy Sci. 45:1487–1492. doi:10.3168/jds.S0022-0302(62)89660-X [Google Scholar]
  18. Li H., Meininger C. J., Bazer F. W., and Wu G.. 2016. Intracellular sources of ornithine for polyamine synthesis in endothelial cells. Amino Acids 48:2401–2410. doi: 10.1007/s00726-016-2256-6 [DOI] [PubMed] [Google Scholar]
  19. NRC 2000. Nutrient requirements of beef cattle. Natl. Acad. Press, Washington, DC. [Google Scholar]
  20. NRC 2001. Nutrient requirements of dairy animals. Natl. Acad. Press, Washington, DC. [Google Scholar]
  21. Owens F. N., and Bergen W. G.. 1983. Nitrogen metabolism of ruminant animals: Historical perspective, current understanding and future implications. J. Anim. Sci. 57(Suppl. 2):498–518. doi:10.2527/animalsci1983. 57Supplement_2498x [PubMed] [Google Scholar]
  22. Scheifinger C., Russell N., and Chalupa W.. 1976. Degradation of amino acids by pure cultures of rumen bacteria. J. Anim. Sci. 43:821–827. doi: 10.2527/jas1976.434821x [DOI] [PubMed] [Google Scholar]
  23. Schwab C. G., Satter L. D., and Clay B.. 1976. Response to lactating dairy cows to abomasal infusion of amino acids. J. Dairy Sci. 59:1254–1270. [DOI] [PubMed] [Google Scholar]
  24. Stalon V., and Mercenier A.. 1984. l -Arginine utilization by Pseudomonas species. J. Gen. Microbiol. 130:69–76. doi: 10.1099/00221287-130-1-69 [DOI] [PubMed] [Google Scholar]
  25. Tagari H., and Bergman E. N.. 1978. Intestinal disappearance and portal blood appearance of amino acids in sheep. J. Nutr. 108:790–803. doi: 10.1093/jn/108.5.790 [DOI] [PubMed] [Google Scholar]
  26. Wei J., Carroll R. J., Harden K. K., and Wu G.. 2012. Comparisons of treatment means when factors do not interact in two-factorial studies. Amino Acids 42:2031–2035. doi: 10.1007/s00726-011-0924-0 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wu G. 1995. Urea synthesis in enterocytes of developing pigs. Biochem. J. 312(Pt 3):717–723. doi: 10.1042/bj3120717 [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wu G. 2013. Amino acids: Biochemistry and nutrition. CRC Press, Boca Raton, FL. [Google Scholar]
  29. Wu G. 2018. Principles of animal nutrition. CRC Press, Boca Raton, FL. [Google Scholar]
  30. Wu G., Bazer F. W., Johnson G. A., and Hou Y. Q.. 2018. Arginine nutrition and metabolism in growing, gestating and lactating swine. J. Anim. Sci. 96:5035–5051. doi:10.1093/jas/sky377 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wu G. Y., and Brosnan J. T.. 1992. Macrophages can convert citrulline into arginine. Biochem. J. 281(Pt 1):45–48. doi: 10.1042/bj2810045 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wu G., Collins J. K., Perkins-Veazie P., Siddiq M., Dolan K. D., Kelly K. A., Heaps C. L., and Meininger C. J.. 2007. Dietary supplementation with watermelon pomace juice enhances arginine availability and ameliorates the metabolic syndrome in zucker diabetic fatty rats. J. Nutr. 137:2680–2685. doi: 10.1093/jn/137.12.2680 [DOI] [PubMed] [Google Scholar]
  33. Wu G., Flynn N. E., and Knabe D. A.. 2000. Enhanced intestinal synthesis of polyamines from proline in cortisol-treated piglets. Am. J. Physiol. Endocrinol. Metab. 279:E395–E402. doi: 10.1152/ajpendo.2000.279.2.E395 [DOI] [PubMed] [Google Scholar]
  34. Wu G., Gilbreath K. R., Bazer F. W., Satterfield M. C., and Cleere J. J.. 2018. Dietary supplementation with an arginine product between Days 1 and 60 of gestation enhances embryonic survival in lactating beef cows. J. Anim. Sci. 96(Suppl. 3):373. doi: 10.1093/jas/sky404.819 [DOI] [Google Scholar]
  35. Wu G., and Meininger C. J.. 2008. Analysis of citrulline, arginine, and methylarginines using high-performance liquid chromatography. Methods Enzymol. 440:177–189. doi: 10.1016/S0076-6879(07)00810-5 [DOI] [PubMed] [Google Scholar]

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