Journal of Animal Science, 2020, 1–9 doi:10.1093/jas/skz370 Advance Access publication December 13, 2019 Received: 2 July 2019 and Accepted: 5 December 2019 Ruminant Nutrition 1 © The Author(s) 2019. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. Ruminant Nutrition Metabolic studies reveal that ruminal microbes of adult steers do not degrade rumen-protected or unprotected l-citrulline Kyler R. Gilbreath, Gayan I. Nawaratna, Tryon A. Wickersham, M. Carey Satterfeld, Fuller W. Bazer, and Guoyao Wu 1 Department of Animal Science, Texas A&M University, College Station, TX 77843 1 Corresponding author: g-wu@tamu.edu ORCiD numbers: 0000-0001-8038-4152 (T. A. Wickersham); 0000-0002-5823-5086 (F. W. Bazer); 0000-0001-8058-6969 (G. Wu). 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 fuid (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 fuid 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 fndings 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. Key words: 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