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. 2019 Dec 13;98(1):skz370. doi: 10.1093/jas/skz370

Metabolic studies reveal that ruminal microbes of adult steers do not degrade rumen-protected or unprotected L-citrulline

Kyler R Gilbreath 1, Gayan I Nawaratna 1, Tryon A Wickersham 1, M Carey Satterfield 1, Fuller W Bazer 1, Guoyao Wu 1,✉
PMCID: PMC6986429  PMID: 31830257

Abstract

In vitro and in vivo experiments were conducted to determine the metabolism of rumen-protected or unprotected l-citrulline (Cit) plus l-glutamine (Gln) by ruminal microbes. In the in vitro experiment, whole ruminal fluid (3 mL, containing microorganisms) from steers was incubated at 37 ºC with 5 mM Cit plus 6 mM Gln (in a rumen-protected or unprotected form) for 0, 0.5, 2, or 4 h after which times 50 µL samples were collected for AA and ammonia analyses. In the in vivo experiment, at 0.5 h before and 0, 0.5, 1, 2, 4, and 6 h after cannulated adult steers consumed 0.56 kg dried-distillers’ grain mixed with 70 g Cit plus 70 g Gln (in a rumen-protected or unprotected form), samples of ruminal fluid and jugular venous blood were obtained for AA analyses. Results from both in vitro and in vivo experiments demonstrated extensive hydrolysis of rumen-unprotected Gln into glutamate, but little degradation of the rumen-protected Gln or rumen-protected and unprotected Cit by ruminal microbes. Concentrations of Cit and arginine in the plasma of steers consuming rumen-protected or unprotected AA increased at 1 and 2 h after the meal, respectively, when compared with values at 0 h. Collectively, these novel findings indicate that ruminal microbes of adult steers do not degrade extracellular Cit in a rumen-protected or unprotected form. Our results refute the view that all dietary AAs are extensively catabolized by ruminal microorganisms and also have important implications for dietary supplementation with Cit to ruminants to enhance the concentration of arginine in their plasma and their productivity.

Keywords: amino acids; arginine, degradation; rumen bacteria

Introduction

l-Citrulline (Cit) and l-glutamine (Gln) play important roles in intestinal health (Blachier et al., 2009; Wu et al., 2011). These two AAs are precursors for the whole-body synthesis of l-arginine in most mammals, including rats, ruminants, swine, and humans (Wu and Morris, 1998; Wu et al., 2018). Oral administration of Cit and Gln to monogastric animals (e.g., rats and swine) increases the concentrations of Cit and arginine in their plasma (Wang et al., 2008; Breuillard et al., 2015; Wu et al., 2018). However, the rates of AA catabolism are high in the rumen, and the microbial population within the rumen has long been considered to extensively degrade dietary AA (Lewis and Emery, 1962; Chalupa, 1976; Schwab et al., 1976; Kung and Rode, 1996; NRC, 2000, 2001; Firkins et al., 2007). Thus, the current view is that dietary-free AA must be protected from degradation in the rumen to enter the small intestine intact (NRC, 2000, 2001).

Based on our 10-yr efforts, we developed a rumen-protected AA (Cit plus Gln) (RPAA) product to feed ruminants (Keith et al., 2018). Our hypothesis for the present study was that dietary supplementation with this product would increase the concentrations of these two AAs in the plasma of ruminants, compared with a rumen-unprotected AA (RUAA) product. To further test the hypothesis, we also conducted in vitro experiments to determine the degradation of Cit and Gln (in a rumen-protected or unprotected form) by microbes in the ruminal fluid of steers. Unexpectedly, our results indicated that ruminal microbes of steers did not catabolize rumen-protected or unprotected extracellular Cit, despite their ability to metabolize rumen-unprotected extracellular Gln. These findings support our recent in vitro observation that ruminal microorganisms do not degrade extracellular-free Cit (Gilbreath et al., 2019) and provide a new, simple method (i.e., dietary supplementation with rumen-unprotected Cit) to increase the concentrations of Cit and arginine in plasma for utilization by various organs and tissues in ruminants.

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.

In Vitro Experiment to Determine the Utilization of Rumen-Protected and Unprotected Cit and Gln by Ruminal Microbes of Steers

Angus steers (~500 kg) had free access to bermudagrass hay (chopped through a 76 x 76 mm wire mesh screen) 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). No cheesecloth was used to prepare ruminal fluid. 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/tube), which contained one of the following: no addition of AA, 3.5 mg RPAA (providing 5 mM rumen-protected Cit plus 6 mM rumen-protected Gln), or 3.5 mg RUAA [providing 5 mM rumen-unprotected Cit plus 6 mM rumen-unprotected Gln). Both products contained Cit, Gln, and binder (soybean hydrogenated oil) in the ratio of 0.25:0.25:0.50 (g:g:g) and were manufactured by Biotechnology Services & Consulting Inc. (Coppell, TX). No carbohydrate or sulfur compounds were added to the incubation medium. Tubes were gassed with CO2 for 20 s, capped and then incubated (70 oscillations/min) at 37 ºC (Gilbreath et al., 2019). At 0, 0.5, 2, and 4 h of the incubation, an aliquot (50 µL) of the medium was collected from the tube without opening the cap. These periods of in vitro incubation were adopted because we found in our preliminary study that the rates of glutamine and arginine catabolism in the ruminal microbes of steers were linear during a 4-h period of incubation, indicating the metabolic viability of the cells.

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. The extracts were stored at −20 ºC until analyzed for AA by high-performance liquid chromatography (HPLC; Wu and Meininger, 2008).

In Vivo Experiment to Determine Changes in Concentrations of AA in Ruminal Fluid and Plasma of Steers Consuming Cit Plus Gln in the Rumen-Protected or Unprotected Form

Sixteen Angus × Hereford steers [538.2 ± 4.1 kg BW] fitted with a ruminal cannula were used in a randomized complete block experiment. Steers (n = 8 per treatment group) were stratified by initial BW and assigned to either the RPAA or the RUAA group. To facilitate consumption, an AA supplement was mixed with dried-distillers’ grains with solubles (DDGS; Table 1) at a 1:2 ratio. The AA supplement in the mixture form was fed at 2% of daily DMI (with Cit and Gln being 0.5% and 0.5% of dietary DM, respectively). Each steer consumed 0.56 kg DDGS plus 0.28 kg of an AA supplement that provided 70 g Cit plus 70 g Gln. The DDGS was selected as a supplement due to its availability, common usage in beef cattle operations, and palatability by all classes of cattle. Throughout the duration of the study, steers were offered bermudagrass hay at 130% of their previous 5-d average of daily feed intake to ensure that access to feed was not limited. The composition of nutrients in the bermudagrass hay is shown in Table 1. All steers had ad libitum access to a trace mineral salt block (≥96% NaCl, 1.00% S, 0.15% Fe, 0.25% Zn, 0.30% Mn, 0.009% I, 0.015% Cu, 0.0025% Co, and 0.001% Se; United Salt Corporation, Houston, TX) and drinking water for the duration of the project. Steers were individually housed for the duration of the project to facilitate determination of individual feed intake. Steers were adapted to their respective AA treatment for 10 d before sampling procedures began and their daily feed intake was determined. Ruminal fluid and blood were sampled on day 15, the final day of the experiment. A 10-d period of adaptation to the supplemental AAs was used because there are reports that ruminal bacteria of steers can adapt well to high-grain diets in 7 d (Fernando et al., 2010), supplemental rumen-degraded intake protein or rumen-undegraded intake protein in 9 d (Latham et al., 2018) or 10 d (Firkins et al., 1986; Bohnert et al., 2002), and various (low to high) levels of feed intake in 10 d (Firkins et al., 1986).

Table 1.

Nutrient composition of bermudagrass hay and dried-distillers’ grains with solubles fed to steers1

Nutrient Bermudagrass hay Dried distillers’ grains with solubles
% of DM
OM 92.1 91.1
Ash 7.9 8.9
NDF 72.3 42.6
 ADF  36.9  19.9
 Hemicellulose  35.4  22.7
Soluble carbohydrates 5.5 15.2
CP 14.3 33.3
 Alanine  1.02 2.10
 Arginine  0.66 1.32
 Asparagine  0.60 1.23
 Aspartate  0.84 1.65
 Cysteine2  0.20 0.57
 Glutamate  1.35 3.37
 Glutamine  0.72 2.58
 Glycine  0.63 1.20
 Histidine  0.28 0.86
 Isoleucine  0.57 1.12
 Leucine  1.06 3.48
 Lysine  0.60 1.02
 Methionine  0.22 0.70
 Phenylalanine  0.67 1.51
 Proline  0.59 2.23
 Serine  0.57 1.47
 Threonine  0.61 1.19
 Tryptophan  0.20 0.26
 Tyrosine  0.43 1.05
 Valine  0.75 1.60
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1OM, ash, NDF, ADF, soluble carbohydrates, and CP were analyzed as described previously (Van Soest, 1967), whereas the content of total AAs (calculated on the basis of their intact molecular weights) was determined using the HPLC method (Hou et al., 2019). Bermudagrass hay and dried-distillers’ grains with solubles contained 87.2% and 90.3% DM, respectively.

2Including cysteine plus 1/2 cystine.

Feeding as well as procedures for sampling blood and ruminal fluid were as follows. At 0600 hours of each day, orts were collected and weighed. Steers received their AA supplements at 0630 hours and the time required to completely consume the supplement was recorded each day, any refusals were collected and weighed after the steers consumed the supplement within 30 min. Bermudagrass hay was fed at 0700 hours during d 0 to 14 of feeding. Steers were checked each afternoon between 1600 and 1800 hours to ensure that they had adequate hay remaining in their bunks. During the final 4 d of the experiment, samples of supplements, hay, and orts were retained for nutrient analysis. Pens were scraped clean every evening starting on day 10 to prevent cross-contamination of fecal samples. Fecal samples were collected during the final 4 d, every 12 h during the sampling period, with the sampling time advancing by 3 h each day, to determine the apparent digestibility of DM in the total gastrointestinal tract (Bell et al., 2017). On day 15, a final BW was recorded, and jugular venous blood (2 mL) and ruminal fluid (20 mL) were collected from each steer at 0.5 h before and 0, 0.5, 1, 2, 4, and 6 h after the animal consumed 0.56 kg DDGS mixed with 70 g Cit plus 70 g Gln in the RPAA or RUAA product; no other feed was offered to the animals between 0600 hours and the end of the 6-h period of blood plus ruminal fluid sampling. Blood and ruminal fluid samples were centrifuged at 600 × g and 10,000 × g for 15 min, respectively, and the supernatant fluid (plasma and microbe-free ruminal fluid, respectively) was collected. An aliquot (100 µL) of the supernatant fluid was acidified with an equal volume of 1.5 M HClO4, followed by the addition of 50 µL of 2 M K2CO3 for neutralization and then 2.25 mL of HPLC-grade water. The extracts were stored at −20 ºC until analyzed for AA by HPLC (Wu and Meininger, 2008; Choi et al., 2014). Ammonia, urea, and glucose were analyzed using enzymatic methods as we described previously (Satterfield et al., 2012, 2013).

Statistical Analysis of Data

Data were analyzed by one- or two-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, as assessed by the Levene’s test (Wei et al., 2012). Differences among treatment means were determined using the Student–Newman–Keuls multiple comparison test. P-value of ≤ 0.05 was taken to indicate statistical significance.

Results and Discussion

In Vitro Experiment

When the RPAA or RUAA product was mixed with microbe-free deionized and double-distilled water for 1 h, analyses of AA in the solution revealed that the concentrations of Cit and Gln in the incubation medium with RPAA were 24.2 ± 0.31% and 24.0 ± 0.37% (mean of ± SEM, n = 10) of values for the RUAA product (Figure 1A and B). Thus, most (~76%), but not all, of Cit or Gln was encapsulated by the binder in the RPAA product.

Figure 1.

Figure 1

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Analysis of free glutamine and citrulline in the rumen-protected AA product (RPAA, Panel A) and rumen-unprotected AA product (RUAA, Panel B). Both products contained l-citrulline (Cit), l-glutamine (Gln), and binder (soybean hydrogenated oil) in the ratio of 0.25:0.25:0.50 (g:g:g). The RPAA or RUAA product (10 g) was mixed with 1 L of deionized and double-distilled water (containing no microorganisms, pH 7.0) for 1 h, and the solution was centrifuged at 600 × g for 10 min. The supernatant fluid was used for the analysis of AAs by HPLC. The concentrations of Cit and Gln in the solution with RPAA were ~24% of values for the RUAA product.

At time 0 h, the concentrations of Cit and Gln in the incubation medium with RPAA were equivalent to ~24% of the values for the RUAA. This result was similar to that obtained when the RPAA was placed in microbe-free water, as noted previously. When ruminal fluid was incubated with RUAA (containing 5 mM rumen-unprotected Cit plus 6 mM rumen-unprotected Gln), Gln was rapidly degraded, with a concomitant increase in the accumulation of glutamate, aspartate, and ammonia in the medium, in a time-dependent manner during the 4-h period of incubation (Table 2). As reported for ruminal protozoa (Onodera et al., 1983), we demonstrated that the mixed ruminal microbes of steers synthesized a very small amount of14Cit (0.11% of catabolized14C-Gln) from 2 mM14C-Gln (Gilbreath et al., 2019). Likewise, a high concentration of glutamate but a low concentration of Cit is present in ruminal bacteria (Leng and Nolan, 1984). This is consistent with our recent finding that ruminal microbes of adult steers extensively hydrolyze extracellular Gln primarily into glutamate but have a limited ability to degrade extracellular glutamate (Gilbreath et al., 2019). In contrast, there was no detectable degradation of rumen-protected or unprotected extracellular Cit by ruminal microbes (Table 2). This is also consistent with our recent report that extracellular 14C-labeled Cit was not degraded by the mixed ruminal microbes of steers (Gilbreath et al., 2019).

Table 2.

AA profile in the incubated ruminal fluid of steers after addition of the AA supplement1

Addition to medium Hour Asp Glu Gln Citrulline Arg Ornithine Ammonia2
None 0 15.0 ± 1.1d 46.8 ± 3.7 33.1 ± 3.4a 5.83 ± 0.75 17.5 ± 2.2 18.4 ± 2.3 0.72 ± 0.08
0.5 19.6 ± 1.3c 49.1 ± 3.5 31.5 ± 3.7a 6.24 ± 0.89 17.1 ± 2.1 21.0 ± 2.6 0.73 ± 0.09
2 24.2 ± 1.6b 48.7 ± 4.2 26.0 ± 3.1b 6.50 ± 0.82 17.6 ± 2.0 22.9 ± 3.0 0.72 ± 0.08
4 30.7 ± 1.9a 53.3 ± 4.8 13.2 ± 1.7c 7.26 ± 0.91 18.8 ± 2.3 22.7 ± 3.2 0.71 ± 0.10
P-value <0.001 0.581 <0.001 0.473 0.812 0.306 0.842
RUAA 0 14.7 ± 1.3d 46.0 ± 1.8d 6027 ± 14a 5011 ± 6 18.3 ± 2.4 18.2 ± 2.1c 0.72 ± 0.09d
0.5 21.5 ± 1.7c 826 ± 45c 5050 ± 39b 5008 ± 22 19.1 ± 2.5 23.9 ± 2.2b 1.46 ± 0.17c
2 28.3 ± 2.0b 1450 ± 63b 3987 ± 171c 5014 ± 24 20.3 ± 2.5 30.5 ± 2.4a 2.13 ± 0.24b
4 34.6 ± 2.4a 2600 ± 178a 2902 ± 233d 5007 ± 10 21.6 ± 2.7 32.8 ± 2.5a 3.20 ± 0.29a
P-value <0.001 <0.001 <0.001 0.997 0.748 <0.001 <0.001
RPAA 0 14.5 ± 1.2d 46.0 ± 1.8d 1468 ± 91a 1207 ± 94 18.2 ± 2.1 18.1 ± 2.0c 0.71 ± 0.08d
0.5 20.3 ± 1.6c 475 ± 85c 984 ± 56b 1213 ± 96 19.4 ± 2.3 21.5 ± 2.2bc 1.05 ± 0.12c
2 26.2 ± 2.0b 709 ± 101b 723 ± 39c 1210 ± 92 19.8 ± 2.0 23.2 ± 2.5ab 1.28 ± 0.14b
4 31.9 ± 2.1a 985 ± 112a 498 ± 47d 1202 ± 95 20.7 ± 2.5 25.4 ± 2.6a 1.62 ± 0.15a
P-value <0.001 <0.001 <0.001 0.985 0.764 0.012 <0.001
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1Values, expressed as nmol/mL of ruminal fluid for AAs and µmol/mL of ruminal fluid for ammonia, are means ± SEM, n = 6.

2The sum of NH3 plus NH4+.

a–dMeans within a column not sharing the same superscript letter differ (P < 0.05), as analyzed by one-way analysis of variance for repeated measures data, followed by the Student–Newman–Keuls multiple comparison test.

RPAA = rumen-protected AA product (5 mM l-citrulline plus 6 mM l-glutamine was added to the incubation medium at time 0 h); RUAA = rumen-unprotected AA product (5 mM l-citrulline plus 6 mM l-glutamine was added to the incubation medium at time hour).

It is generally recognized that dietary AAs are extensively degraded by ruminal bacteria (Firkins et al., 2007; Wallace et al., 1997; NRC, 2000, 2001).To date, there is little evidence that all AAs are degraded by ruminal bacteria. However, some authors believe otherwise. For example, Owens and Basalan (2016) stated that “all amino acids that are not linked or guarded from attack are extensively degraded in the rumen to ammonia, carbon dioxide, VFA, and branched-chain fatty acids.” Likewise, Tedeschi and Fox (2016) concluded that “negligible, if any, amounts of amino-N (peptide and free AA) would escape the rumen.” We observed that there was no uptake of 14C-Cit by the ruminal microbes of steers (Gilbreath et al., 2019). Clearly, results of our in vitro experiments indicate that these cells did not catabolize extracellular Cit, which is abundant in some plants such as watermelon (Wu, 2013).

In Vivo Experiment

During d 0 to 14 of the experimental period, intakes of bermudagrass hay by steers in the RUAA and RPAA groups were 14.02 ± 0.33 and 14.04 ± 0.35 kg DM/d per steer (mean ± SEM, n = 8), respectively (P > 0.05). The apparent digestibility (%) of DM in the total gastrointestinal tract of steers was 73.2 ± 2.6 and 73.4 ± 2.7 (mean ± SEM, n = 8), respectively, in the RUAA and RPAA groups (P > 0.05). Thus, the RPAA supplement did not affect the feed intake or DM digestion of steers. In all steers, the concentrations of free AA in the ruminal fluid were very low ranging from 1.3 nmol/mL for taurine to 44 nmol/mL for glutamate (Table 3). This is due to a number of factors, such as a limited amount of free AA in the diet, active degradation of dietary protein-derived AA by ruminal bacteria (via transaminases, dehydrogenases, and deaminases), rapid microbial uptake of peptides and free AA, as well as the constant flow of AA-containing ruminal fluid into other parts of the forestomach (Wallace et al., 1997).

Table 3.

Concentrations of AAs and ammonia in the ruminal fluid of steers after their consumption of AA supplement1

AA RUAA RPAA P-values
0 h 0.5 h 1 h 2 h 4 h 6 h 0 h 0.5 h 1 h 2 h 4 h 6 h AA Time AA × time
Asp 16 ± 0.9 26 ± 5.0 35 ± 7.3 20 ± 1.3 18 ± 1.0 15 ± 0.9 16 ± 1.2 16 ± 1.4 16 ± 1.6 15 ± 1.4 16 ± 1.7 16 ± 2.1 0.062 <0.001 0.023
Glu 44 ± 2.8 338 ± 48 523 ± 45 243 ± 22 29 ± 2.2 32 ± 4.4 47 ± 1.0 46 ± 2.3 48 ± 3.1 46 ± 0.9 39 ± 2.1 46 ± 1.7 <0.001 <0.001 <0.001
Asn 6.2 ± 0.4 6.8 ± 0.3 6.4 ± 0.4 6.6 ± 0.5 7.2 ± 0.4 6.9 ± 0.2 6.4 ± 0.2 6.3 ± 0.3 6.7 ± 0.3 6.6 ± 0.1 6.4 ± 0.1 6.3 ± 0.4 0.258 0.681 0.406
Ser 17 ± 2.3 25 ± 2.0 32 ± 4.2 27 ± 3.8 23 ± 7.1 18 ± 4.1 18 ± 0.9 19 ± 1.2 18 ± 1.4 15 ± 1.6 15 ± 2.1 14 ± 1.8 0.072 <0.001 0.015
Gln 31 ± 1.3 3570 ± 887 2108 ± 432 228 ± 80 102 ± 29 35 ± 4.9 34 ± 2.8 120 ± 26 61 ± 27 32 ± 2.5 28 ± 2.6 30 ± 3.7 0.001 <0.001 <0.001
His 8.4 ± 0.9 14 ± 2.3 15 ± 1.9 11 ± 1.3 10 ± 2.3 8.8 ± 1.7 7.7 ± 0.7 8.4 ± 0.7 8.2 ± 0.9 8.6 ± 1.1 8.0 ± 0.7 7.9 ± 1.0 0.002 <0.001 <0.001
Gly 25 ± 3.5 27 ± 3.3 28 ± 4.5 26 ± 4.8 26 ± 3.5 23 ± 3.9 26 ± 3.4 29 ± 2.0 30 ± 2.0 28 ± 1.2 28 ± 2.1 26 ± 1.3 0.420 0.009 0.529
Thr 14 ± 2.1 15 ± 2.5 15 ± 2.5 15 ± 2.9 14 ± 2.3 14 ± 2.5 17 ± 2.4 16 ± 2.8 15 ± 1.8 15 ± 2.0 15 ± 2.2 15 ± 1.4 0.641 0.096 0.166
Cit 5.4 ± 0.4 4481±1095 5436±1187 2800 ± 439 761 ± 389 20 ± 6.9 6.2 ± 0.7 575 ± 62 693 ± 85 513 ± 94 106 ± 63 6.6 ± 1.1 <0.001 <0.001 <0.001
Arg 20 ± 2.2 32 ± 2.4 37 ± 3.8 26 ± 4.5 20 ± 5.7 18 ± 5.4 21 ± 3.0 21 ± 3.0 22 ± 2.0 22 ± 3.3 21 ± 3.5 20 ± 2.6 0.244 <0.001 0.002
β-Ala 12 ± 1.1 20 ± 3.0 26 ± 7.7 21 ± 3.0 19 ± 2.7 14 ± 1.1 11 ± 1.2 11 ± 1.2 14 ± 3.1 15 ± 3.5 13 ± 2.8 12 ± 2.4 0.092 <0.001 0.002
Tau 1.3 ± 0.2 1.4 ± 0.1 1.3 ± 0.1 1.3 ± 0.1 1.3 ± 0.1 1.4 ± 0.1 1.4 ± 0.1 1.3 ± 0.1 1.2 ± 0.1 1.3 ± 0.1 1.1 ± 0.1 1.2 ± 0.1 0.369 0.324 0.716
Ala 17 ± 1.9 48 ± 9.3 56 ± 10 33 ± 10 26 ± 7.1 22 ± 5.7 16 ± 2.4 16 ± 1.8 15 ± 1.5 15 ± 1.3 15 ± 1.6 14 ± 1.8 0.028 <0.001 <0.001
Tyr 12 ± 1.4 14 ± 1.7 12 ± 1.7 13 ± 1.5 12 ± 1.4 12 ± 1.0 11 ± 1.5 12 ± 1.7 12 ± 2.4 12 ± 2.2 11 ± 2.1 10 ± 2.5 0.614 <0.001 <0.001
Trp 8.7 ± 1.0 8.0 ± 0.9 8.0 ± 0.6 8.7 ± 1.1 8.4 ± 1.1 8.2 ± 1.5 8.7 ± 0.8 8.6 ± 0.7 8.1 ± 0.7 8.3 ± 0.7 8.2 ± 0.4 7.7 ± 0.4 0.950 0.087 0.681
Met 7.3 ± 0.8 7.4 ± 0.9 7.2 ± 1.1 6.9 ± 0.8 6.9 ± 0.8 6.6 ± 0.7 7.1 ± 0.4 7.1 ± 0.6 7.0 ± 0.9 6.9 ± 0.7 6.6 ± 0.5 6.5 ± 0.6 0.882 0.009 0.718
Val 11 ± 1.4 11 ± 1.0 11 ± 1.5 9.5 ± 1.5 9.8 ± 1.2 10 ± 0.8 9.8 ± 0.6 11 ± 1.0 9.5 ± 0.7 8.8 ± 0.6 8.1 ± 0.4 9.0 ± 0.7 0.661 0.007 0.030
Phe 7.2 ± 0.8 7.6 ± 0.8 7.5 ± 0.9 7.4 ± 0.5 8.0 ± 0.8 8.0 ± 0.8 7.8 ± 0.5 8.7 ± 0.4 8.4 ± 1.1 8.0 ± 0.6 7.7 ± 0.4 7.4 ± 0.4 0.645 0.341 0.032
Ile 9.5 ± 1.6 10 ± 1.4 9.6 ± 1.4 9.3 ± 1.7 9.1 ± 1.7 8.2 ± 1.2 8.6 ± 0.5 9.1 ± 0.5 9.9 ± 0.4 8.9 ± 0.5 8.3 ± 0.4 8.5 ± 0.7 0.969 0.021 0.196
Leu 14 ± 1.3 15 ± 1.9 15 ± 1.7 13 ± 1.8 13 ± 1.8 12 ± 1.5 13 ± 1.7 13 ± 2.1 13 ± 1.5 12 ± 1.6 13 ± 2.2 12 ± 1.9 0.603 <0.001 0.199
Orn 11 ± 1.7 27 ± 6.3 49 ± 4.1 20 ± 3.4 13 ± 1.5 14 ± 2.0 12 ± 3.2 13 ± 3.4 12 ± 2.6 11 ± 2.7 12 ± 2.9 11 ± 3.0 0.045 <0.001 <0.001
Lys 10 ± 1.9 10 ± 2.1 10 ± 2.0 9.7 ± 1.8 9.4 ± 1.6 9.5 ± 1.7 9.9 ± 1.7 9.6 ± 1.1 10 ± 1.4 9.4 ± 1.1 9.3 ± 1.6 9.5 ± 2.0 0.943 0.480 0.857
Pro 14 ± 1.0 21 ± 1.5 35 ± 1.9 19 ± 1.3 17 ± 1.1 15 ± 1.3 13 ± 1.2 14 ± 1.0 14 ± 1.3 15 ± 1.7 13 ± 1.4 12 ± 1.4 0.039 0.027 0.038
Cys 6.2 ± 0.5 6.5 ± 0.6 6.4 ± 0.6 6.8 ± 0.8 6.3 ± 0.5 6.1 ± 0.6 6.5 ± 0.6 6.8 ± 0.8 6.7 ± 0.6 6.3 ± 0.5 6.2 ± 0.7 6.0 ± 0.8 0.922 0.854 0.761
NH3 0.84 ± 0.06 1.21 ± 0.05 1.45 ± 0.09 1.06 ± 0.06 0.83 ± 0.07 0.85 ± 0.06 0.86±0.06 0.90±0.06 0.92±0.06 0.87±0.06 0.85±0.06 0.83±0.07 0.041 <0.018 0.035
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1Values, expressed as nmol/mL of ruminal fluid for AAs and µmol/mL of ruminal fluid for ammonia, are means ± SEM, n = 8. Results were analyzed by two-way analysis of variance for repeated measures data.

NH3 = sum of NH3 plus NH4+; Orn = ornithine; Tau = taurine; RPAA = rumen-protected AA product; RUAA = rumen-unprotected AA product.

In RUAA or RPAA steers, concentrations of all measured free AAs in ruminal fluid did not differ (P > 0.05) between 30 and 0 min before DDGS feeding. Concentrations of free AA in the ruminal fluid of steers after their consumption of either the RPAA or RUAA supplement are summarized in Table 3. Glutamate was the most abundant AA in ruminal fluid obtained from both groups of steers, followed by Gln, glycine, arginine, alanine, and serine in order of decreasing abundance. Concentrations of asparagine, threonine, taurine, tryptophan, phenylalanine, lysine, and total cysteine in ruminal fluid did not differ (P > 0.05) throughout the 6-h sampling period in either group of steers. However, concentrations of many AAs (including Cit, Gln, arginine, ornithine, and proline) in ruminal fluid showed time-dependent changes (P < 0.05), indicating that oral administration of RUAA or RPAA increased their concentrations in the rumen. Of particular note, consistent with the active formation of 14C-glutamate from 14C-Gln by the mixed ruminal microbes of steers (Gilbreath et al., 2019), a large amount of glutamate was produced from rumen-unprotected Gln and accumulated in the rumen of steers (Table 3). Dietary and rumen-derived glutamate can enter the small intestine for subsequent utilization, and this aspect of glutamate metabolism should be quantified in future studies involving ruminants with a fitted cannula in the duodenum. In ruminants [including sheep (Tagari and Bergman, 1978) and cattle (Reynolds, 2006)], as in nonruminants [such as pigs and rats (Wu, 1998; Blachier et al., 2009; Burrin and Stoll, 2009)], nearly all glutamate (95% to 97%) in the lumen of the small intestine is metabolized during the first pass into the portal vein. Major products of intestinal glutamate catabolism include CO2, alanine, aspartate, ornithine, Cit, arginine, proline, and glutathione (Wu, 1998; Blachier et al., 2009; Hou and Wu, 2018). Additionally, through oxidation via the Krebs cycle, glutamate is a major metabolic fuel for mammalian and avian enterocytes (Wu, 2018). The fact that the patterns of AA in ruminal fluid (Table 3) differed from those in the plasma of steers (Table 4) reflects, in part, the extensive metabolism of diet- and microbial protein-derived AA in the small intestine.

Table 4.

Concentrations of AAs, ammonia, urea, and glucose in the plasma of steers after their consumption of AA supplement1

AA RUAA RPAA P-values
0 h 0.5 h 1 h 2 h 4 h 6 h 0 h 0.5 h 1 h 2 h 4 h 6 h AA Time AA × time
Asp 5.4 ± 0.5 4.3 ± 0.5 3.9 ± 0.5 4.0 ± 0.5 4.4 ± 0.4 3.9 ± 0.5 4.0 ± 0.4 3.7 ± 0.4 3.7 ± 0.3 4.0 ± 0.5 4.7 ± 1.7 4.4 ± 1.6 0.696 0.092 0.235
Glu 52 ± 4.2 47 ± 1.8 43 ± 2.5 45 ± 3.3 43 ± 4.0 40 ± 2.2 47 ± 3.5 50 ± 2.5 43 ± 3.9 42 ± 2.8 46 ± 15 43 ± 14 0.937 0.278 0.841
Asn 31 ± 2.1 29 ± 1.6 28 ± 1.4 25 ± 1.5 23 ± 0.8 22 ± 1.5 27 ± 2.5 28 ± 1.8 28 ± 2.3 26 ± 1.8 26 ± 9.3 23 ± 8.3 0.933 <0.001 0.162
Ser 67 ± 2.3 70 ± 3.6 68 ± 4.2 59 ± 3.3 57 ± 2.9 59 ± 3.4 60 ± 2.9 62 ± 3.1 63 ± 4.6 62 ± 3.6 62 ± 2.2 58 ± 2.0 0.105 0.001 0.105
Gln 286 ± 17 294 ± 18 311 ± 22 311 ± 19 296 ± 17 289 ± 17 268 ± 18 262 ± 14 277 ± 17 274 ± 15 302 ± 20 288 ± 17 0.432 0.003 0.090
His 67 ± 5.2 66 ± 5.3 64 ± 6.1 63 ± 4.9 57 ± 4.9 56 ± 4.8 56 ± 5.8 55 ± 5.5 57 ± 4.7 56 ± 4.9 57 ± 19 53 ±18 0.378 0.036 0.112
Gly 347 ± 25 345 ± 30 344 ± 32 339 ± 22 356 ± 25 342 ± 36 325 ± 28 347 ± 33 354 ± 42 345 ± 36 378 ± 26 370 ± 34 0.866 0.366 0.602
Thr 62 ± 3.0 64 ± 4.8 64 ± 5.9 61 ± 5.6 56 ± 4.3 53 ± 5.7 60 ± 6.4 64 ± 6.3 68 ± 5.4 61 ± 6.1 62 ± 2.1 56 ± 1.9 0.781 0.015 0.797
Cit 87 ± 1.8 94 ± 3.7 101 ± 5.2 102 ± 4.3 106 ± 3.5 97 ± 2.7 97 ± 5.6 103 ± 6.4 112 ± 6.6 113 ± 5.3 117 ± 4.8 107 ± 7.0 0.112 <0.001 0.978
Arg 121 ± 5.1 131 ± 6.3 133 ± 9.5 134 ± 6.4 138 ± 5.9 126 ± 4.9 122 ± 6.2 131 ± 6.3 141 ± 7.2 146 ± 8.9 156 ± 5.4 134 ± 5.3 0.363 <0.001 0.064
β-Ala 12 ± 2.1 12 ± 2.4 14 ± 2.5 15 ± 2.3 17 ± 2.4 16 ± 2.8 12 ± 1.2 12 ± 1.1 13 ± 1.8 14 ± 1.7 15 ± 5.1 15 ± 5.1 0.791 <0.001 0.939
Tau 39 ± 5.9 29 ± 3.8 26 ± 3.9 29 ± 3.9 28 ± 3.2 28 ± 3.5 27 ± 2.0 28 ± 2.6 25 ± 3.5 26 ± 2.9 33 ± 11 29 ± 9.5 0.633 0.108 0.081
Ala 181 ± 10 184 ± 13 190 ± 12 241 ± 25 169 ± 8.3 168 ± 6.6 164 ± 6.9 158 ± 8.8 169 ± 12 180 ± 16 164 ± 10 169 ± 9.2 0.168 <0.001 0.025
Tyr 70 ± 3.0 67 ± 2.6 66 ± 3.0 64 ± 2.9 55 ± 2.0 53 ± 1.5 65 ± 3.4 66 ± 2.8 65 ± 3.0 62 ± 2.2 58 ± 19 54 ± 18 0.847 <0.001 0.635
Trp 49 ± 3.2 47 ± 3.1 49 ± 3.0 50 ± 3.6 47 ± 4.0 48 ± 4.5 48 ± 3.8 50 ± 6.3 54 ± 5.1 52 ± 5.5 52 ± 17 48 ± 16 0.701 0.555 0.582
Met 27 ± 0.8 27 ± 0.8 27 ± 1.4 26 ± 1.0 26 ± 1.1 25 ± 1.2 25 ± 2.0 26 ± 1.7 27 ± 1.4 26 ± 1.3 26 ± 0.9 26 ± 0.9 0.872 0.701 0.701
Val 224 ± 11 221 ± 9.1 215 ± 9.8 207 ± 9.6 180 ± 6.7 171 ± 6.5 196 ± 12 206 ± 8.7 210 ± 12 201 ± 8.2 185 ± 6.2 178 ± 5.9 0.520 <0.001 0.238
Phe 51 ± 2.6 52 ± 2.5 52 ± 2.6 51 ± 2.5 44 ± 1.4 43 ± 2.2 49 ± 1.4 52 ± 1.9 53 ± 3.6 52 ± 2.2 49 ± 16 45 ± 15 0.643 <0.001 0.426
Ile 100 ± 4.9 95 ± 3.3 92 ± 4.4 83 ± 3.1 71 ± 2.4 67 ± 2.3 87 ± 5.3 90 ± 3.5 87 ± 5.6 84 ± 4.2 81 ± 2.7 74 ± 2.5 0.860 <0.001 0.026
Leu 148 ± 6.9 141 ± 6.4 138 ± 6.5 128 ± 5.7 110 ± 5.6 104 ± 5.3 130 ± 8.2 136 ± 6.2 132 ± 7.5 129 ± 5.2 122 ± 11 111 ± 10 0.855 <0.001 0.037
Orn 106 ± 8.0 95 ± 5.6 99 ± 7.0 88 ± 5.7 89 ± 6.3 83 ± 6.0 103 ± 10 101 ± 8.0 99 ± 7.8 95 ± 8.0 93 ± 8.2 92 ± 8.8 0.717 0.012 0.424
Lys 104 ± 5.8 96 ± 5.5 98 ± 8.8 83 ± 4.4 77 ± 5.1 70 ± 5.6 87 ± 10 88 ± 7.8 90 ± 3.3 81 ± 5.7 73 ± 7.0 72 ± 6.6 0.885 <0.001 0.586
Pro 184 ± 11 186 ± 12 189 ± 9.2 181 ± 5.6 178 ± 5.6 175 ± 5.7 186 ± 9.5 190 ± 7.4 189 ± 9.1 182 ± 6.9 180 ± 6.7 177 ± 8.5 0.713 0.248 0.519
Cys 132 ± 4.1 135 ± 11 140 ± 8.0 135 ± 6.6 130 ± 7.2 133 ± 6.5 134 ± 8.3 140 ± 9.6 143 ± 6.7 136 ± 7.5 129 ± 7.1 131 ± 10 0.604 0.197 0.442
NH3 86 ± 4.7 92 ± 6.6 102 ± 5.9 98 ± 4.9 93 ± 4.5 87 ± 5.1 84 ± 4.8 87 ± 5.0 95 ± 3.5 92 ± 6.6 87 ± 4.8 85 ± 5.5 0.674 0.154 0.706
Urea 6.08±0.35 6.22±0.29 6.38±0.32 6.70±0.20 6.34±0.12 6.52±0.15 6.16±0.23 6.34±0.19 6.70±0.19 6.68±0.18 6.24±0.31 6.06±0.24 0.559 0.183 0.624
Gluc 3.42±0.12 3.58±0.13 3.72±0.15 3.62±0.18 3.54±0.12 3.46±0.14 3.52±0.13 3.60±0.10 3.84±0.14 3.70±0.16 3.65±0.10 3.56±0.09 0.682 0.136 0.572
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1Values, expressed as nmol/mL for AAs and ammonia and as µmol/mL for glucose and urea, are means ± SEM, n = 8. Results were analyzed by two-way analysis of variance for repeated measures data.

Gluc = glucose; NH3 = sum of NH3 plus NH4+; Orn = ornithine; Tau = taurine. RPAA = rumen-protected AA product; RUAA = rumen-unprotected AA product.

In the RUAA group, Gln and Cit were released rapidly from the RUAA product to increase their concentrations in ruminal fluid, with values for Gln and Cit peaking at 0.5 and 1 h, respectively (Table 3). This is likely because of their different rates of AA turnover in the rumen and/or different rates of utilization by ruminal microbes. In the rumen of steers fed the RUAA product, Gln underwent extensive catabolism to generate ammonia, glutamate, serine, arginine, alanine, ornithine, and proline. Concentrations of both Cit and Gln in the ruminal fluid of RUAA steers declined rapidly after 1 h, likely because of a rapid flow of the AA out of the rumen into the other compartments of the forestomach and, in the case of Gln, a high rate of degradation by ruminal microbes as noted previously. In our preliminary studies, we found that feeding a small amount of DDGS (0.56 kg) to 6 steers did not significantly affect the concentrations of free glutamate (41 ± 2.9 to 47 ± 3.5 µM), Gln (32 ± 1.8 to 36 ± 2.2 µM), Cit (5.6 ± 0.54 to 6.3 ± 0.59 µM) or any other AA in ruminal fluid during a 6-h period postfeeding (K. R. Gilbreath and G. Wu, unpublished observations). Thus, the concentrations of free AA in the ruminal fluid of steers were unaffected by feeding a small amount of DDGS without any AA supplement. Importantly, the 11-, 67-, and 1,006-fold increases in the concentrations of glutamate, Gln, and Cit, respectively, in the ruminal fluid of steers at 1 h post-RUAA feeding (Table 3) resulted from the RUAA supplementation rather than the basal metabolism of the ruminal bacteria exposed to very low concentrations of free AAs.

The magnitudes of changes in the concentrations of Cit and Gln in the ruminal fluid of RPAA steers were much less than those for RUAA steers (Table 3), indicating that a majority of Cit and Gln in the RPAA product was not released into the rumen. This result is consistent with the in vitro data indicating that most (~76%) of the Gln and Cit in the RPAA product was encapsulated by the binder (Figure 1). Interestingly, at times 0.5, 1, 2, and 4 h, the concentrations of Cit in the ruminal fluid of the RPAA steers were only 12.8%, 12.7%, 18.3%, and 13.9%, respectively, of those in the RUAA steers, possibly because the RPAA product was more stable in the rumen in vivo than in deionized-distilled water in vitro. An alternative explanation is that as the concentration of Cit in the ruminal fluid of RPAA steers was much lower than that for RUAA steers, a greater percentage of Cit exited the rumen in the RPAA steers compared with the RUAA steers. Interestingly, at 0.5 and 1 h, the concentrations of Gln in the ruminal fluid of the RPAA steers were only about 3% of those in the RUAA steers (Table 3). We suggest that the small amount of Gln released from the RPAA product within the rumen was rapidly metabolized by its microbe, therefore resulting in low concentrations of Gln in ruminal fluid during a 6-h period. Due to the limitations of timeframes (0, 0.5, 1, 2, 4, and 6 h) for sampling ruminal fluid from steers, rumen passage rates of Gln, Cit, and other AAs could not be measured in our experiments. Detailed kinetic studies are needed to address this issue.

In RUAA or RPAA steers, concentrations of all measured free AAs in plasma did not differ (P > 0.05) between 30 and 0 min befor DDGS feeding. Concentrations of free AAs in the plasma of steers after their consumption of either the RPAA or RUAA supplement are summarized in Table 4. Glycine was the most abundant AA in plasma obtained from both groups of steers, followed by Gln, valine, proline, alanine, leucine, total cysteine, lysine, and Cit in order of decreasing abundance. Concentrations of aspartate, glutamate, glycine, tryptophan, methionine, proline, total cysteine, ammonia, urea, and glucose in plasma did not differ (P > 0.05) throughout the 6-h sampling period in either group. However, concentrations of other AA (including Cit, Gln, arginine, and ornithine) in plasma showed time-dependent increases in response to oral administration of RUAA or RPAA, likely due to increases in their endogenous syntheses. Glutamine in the rumen of the RUAA group could be used by its microbes for the syntheses of AA (including glutamate, aspartate, and threonine, which are all precursors of branched-chain AA) and then proteins (Owens and Bergen, 1983; Firkins et al., 2007; Wu, 2013). Microbial proteins would then be available for digestion in the abomasum and the small intestine to release Gln and other AA (including branched-chain AA). Primarily in skeletal muscle, branched-chain AA transaminate with α-ketoglutarate to form glutamate, which is amidated with ammonia by Gln synthetase to generate Gln (Wu, 2013). In the RPAA group, the rumen-protected Gln that escaped the rumen could either enter the portal vein or be utilized by enterocytes in the small intestine to synthesize Cit (Wu and Morris, 1998). In both the RUAA and RPAA groups, Cit could escape the rumen to enter the small intestine for absorption into the portal vein and then into the systemic blood circulation (Wu, 2018). Cit in the blood is utilized for the synthesis of arginine, which is then converted into ornithine primarily via arginase and, to a lesser extent, via arginine:glycine amidinotransferase (Wu and Morris, 1998; Wu et al., 2018). Thus, as shown in Table 4, dietary supplementation with Cit in either the rumen-protected or nonprotected form can effectively increase the circulating levels of both Cit and arginine [an AA that can improve lactation, fetal growth and development, and meat quality of ruminants (Choi et al., 2014; Sales et al., 2016; Reynolds et al., 2019; Teixeira et al., 2019)]. This finding is important for the nutrition of ruminants, because it indicates that there is no need to protect Cit when this AA is supplemented to their diets. Because Cit is effectively converted into arginine in ruminants and has a longer half-life than arginine in their blood (Lassala et al., 2009), expensive and time-consuming procedures for encapsulating Cit or arginine as a means of providing enteral arginine to ruminants are not required.

In conclusion, the results of our experiments indicated that, in contrast to Gln, neither rumen-protected nor rumen-unprotected Cit is catabolized by in vitro ruminal microorganisms from adult steers. Dietary supplementation with either rumen-protected or rumen-unprotected Cit plus Gln to steers increased both Cit and arginine in their plasma. These findings correct the view that all dietary AAs are degraded extensively by ruminal microbes. Therefore, Cit, without encapsulation or protection from ruminal microbes, can be used as an effective additive to the diets of ruminants to enhance arginine availability in their blood and other tissues for metabolic utilization, as well as their productivity (e.g., growth, lactation, and reproduction). Future studies are warranted to determine the rumen passage rates of Gln, Cit, and other AAs in adult steers.

Funding

Supported by the Texas A&M AgriLife Research Beef Program and the Animal Science Mini-Grant Program. We thank W. David Long, Neil D. Wu, and Shengdi Hu for technical assistance, as well as Unkyung Lee for help with statistical analyses of data.

Conflict of interest statement

None declared.

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