L-Arginine Nutrition and Metabolism in Ruminants.

L-Arginine (Arg) plays a central role in the nitrogen metabolism (e.g., syntheses of protein, nitric oxide, polyamines, and creatine), blood flow, nutrient utilization, and health of ruminants. This amino acid is produced by ruminal bacteria and is also synthesized from L-glutamine, L-glutamate, and L-proline via the formation of L-citrulline (Cit) in the enterocytes of young and adult ruminants. In pre-weaning ruminants, most of the Cit formed de novo by the enterocytes is used locally for Arg production. In post-weaning ruminants, the small intestine-derived Cit is converted into Arg primarily in the kidneys and, to a lesser extent, in endothelial cells, macrophages, and other cell types. Under normal feeding conditions, Arg synthesis contributes 65% and 68% of total Arg requirements for nonpregnant and late pregnany ewes fed a diet with ~12% crude protein, respectively, whereas creatine production requires 40% and 36% of Arg utilized by nonpregnant and late pregnant ewes, respectively. Arg has not traditionally been considered a limiting nutrient in diets for post-weaning, gestating, or lactating ruminants because it has been assumed that these animals can synthesize sufficient Arg to meet their nutritional and physiological needs. This lack of a full understanding of Arg nutrition and metabolism has contributed to suboptimal efficiencies for milk production, reproductive performance, and growth in ruminants. There is now considerable evidence that dietary supplementation with rumen-protected Arg (e.g., 0.25-0.5% of dietary dry matter) can improve all these production indices without adverse effects on metabolism or health. Because extracellular Cit is not degraded by microbes in the rumen due to the lack of uptake, Cit can be used without any encapsulation as an effective dietary source for the synthesis of Arg in ruminants, including dairy and beef cows, as well as sheep and goats. Thus, an adequate amount of supplemental rumen-protected Arg or unencapsulated Cit is necessary to support maximum survival, growth, lactation, reproductive performance, and feed efficiency, as well as optimum health and well-being in all ruminants.

Amino Acid Nutrition and Reproductive Performance in Ruminants.

Amino acids (AAs) are essential for the survival, growth and development of ruminant conceptuses. Most of the dietary AAs (including L-arginine, L-lysine, L-methionine and L-glutamine) are extensively catabolized by the ruminal microbes of ruminants to synthesize AAs and microbial proteins (the major source of AAs utilized by cells in ruminant species) in the presence of sufficient carbohydrates (mainly cellulose and hemicellulose), nitrogen, and sulfur. Results of recent studies indicate that the ruminal microbes of adult steers and sheep do not degrade extracellular L-citrulline and have a limited ability to metabolize extracellular L-glutamate due to little or no uptake by the cells. Although traditional research in ruminant protein nutrition has focused on AAs (e.g., lysine and methionine for lactating cows) that are not synthesized by eukaryotic cells, there is growing interest in the nutritional and physiological roles of AAs (e.g., L-arginine, L-citrulline, L-glutamine and L-glutamate) in gestating ruminants (e.g., cattle, sheep and goats) and lactating dairy cows. Results of recent studies show that intravenous administration of L-arginine to underfed, overweight or prolific ewes enhances fetal growth, the development of brown fat in fetuses, and the survival of neonatal lambs. Likewise, dietary supplementation with either rumen-protected L-arginine or unprotected L-citrulline to gestating sheep or beef cattle improved embryonic survival. Because dietary L-citrulline and L-glutamate are not degraded by ruminal microbes, addition of these two amino acids may be a new useful, cost-effective method for improving the reproductive efficiency of ruminants.

L-Arginine promotes protein synthesis and cell growth in brown adipocyte precursor cells via the mTOR signal pathway.

L-Arginine has been reported to enhance brown adipose tissue developments in fetal lambs of obese ewes, but the underlying mechanism is unknown. The present study tested the hypothesis that L-arginine stimulates growth and development of brown adipocyte precursor cells (BAPCs) through activation of mammalian target of rapamycin cell signaling. BAPCs isolated from fetal lambs at day 90 of gestation were incubated   for 6 h in arginine-free DMEM, and then cultured in DMEM with concentrations of 50, 100, 200, 500 or 1000 µmol L-arginine/L for 24-96 h. Cell proliferation, protein turnover, the mammalian target of rapamycin (mTOR) signaling pathway and pre-adipocyte differentiation markers were determined. L-arginine treatment enhanced (P < 0.05) BAPC growth and protein synthesis, while inhibiting proteolysis in a dose-dependent manner. Compared with 50 and 100 µmol/L (the concentrations of arginine in the maternal plasma of obese ewes), 200 µmol L-arginine/L (the concentrations of arginine in the maternal plasma of obese ewes receiving arginine supplementation) increased (P < 0.05) the abundances of phosphorylated mTOR, P70S6K and 4EBP1, as well as the abundances of PGC1α, UCP1, BMP7 and PRDM16. These novel findings indicate that increasing extra-cellular arginine concentration from 50 to 200 µmol/L activates mTOR cell signaling in BAPCs and enhances their growth and development in a dose-dependent manner. Our results provide a mechanism for arginine supplementation to enhance the development of brown adipose tissue in fetal lambs.

Ruminal microbes of adult sheep do not degrade extracellular l-citrulline.

This study determined whether extracellular citrulline is degraded by ruminal bacteria of sheep. In the first experiment, whole rumen fluid (3 mL) from six adult Suffolk sheep was incubated at 37 °C with 5 mM l-glutamine (Gln), l-glutamate (Glu), l-arginine (Arg), or l-citrulline (Cit) for 0, 0.5, 1, and 2 h or with 0, 0.5, 2, or 5 mM Gln, Glu, Arg, or Cit for 2 h. An aliquot (50 µL) of the incubation solution was collected at the predetermined time points for amino acids (AA) analyses. Results showed extensive hydrolysis of Gln into Glu and ammonia, of Arg into l-ornithine and l-proline, but little or no degradation of extracellular Cit or Glu by ruminal microbes. In the second experiment, six adult Suffolk sheep were individually fed each of three separate supplements (8 g Gln , Cit, or urea) on three separate days along with regular feed (800 g/animal). Blood (2 mL) was sampled from the jugular vein prior to feeding (time 0) and at 0.5, 1, 2, and 4 h after consuming the supplement. Plasma was analyzed for AA, glucose, ammonia, and urea. The concentrations of Cit in the plasma of sheep consuming this AA increased (P < 0.001) by 117% at 4 h and those of Arg increased by 23% at 4 h, compared with the baseline values. Urea or Gln feeding did not affect (P > 0.05) the concentrations of Cit or Arg in plasma. These results indicate that Cit is not metabolized by ruminal microbes of sheep and is, therefore, absorbed as such by the small intestine and used for the synthesis of Arg by extrahepatic tissues.

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

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.

Analysis of repeated measures data in nutrition research.

Amino acid nutrition studies often involve repeated measures data. An example is that the concentrations of plasma citrulline in steers are repeatedly measured from the same animals. The standard repeated measures ANOVA method does not detect significant time changes in the concentrations of plasma citrulline within 6 hours after steers consumed rumen-protected citrulline, while a graphical analysis indicates that there exists a time effect. Here we describe three mixed model analyses that capture the time effect in a statistically significant way, while accounting for the correlations of measurements over time from the same steers. First, we allow flexible variance-covariance structures on our model. Second, we use baseline measurements as a covariate in our model. Third, we use percent-change from baseline as a data normalization method. In our data analysis, all these three approaches can lead to meaningful statistical results that oral administration of rumen-protected citrulline enhances the concentrations of plasma citrulline over time in ruminants. This supports the notion that rumen-protected citrulline can bypass the rumen to effectively enter the blood circulation.

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

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.