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Title: METABOLIC FATES OF AMMONIA NITROGEN IN RUMINAL EPITHELIAL AND DUODENAL MUCOSAL CELLS ISOLATED FROM GROWING SHEEP.

Author
item OBA, MASAHITO - UNIVERSITY OF MARYALND
item Baldwin, Ransom - Randy
item OWENS, SANDRA - UNIVERSITY OF MARYLA
item BEQUETTE, BRIAN - UNIVERSITY OF MARYLAND

Submitted to: Journal of Dairy Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/3/2005
Publication Date: 11/15/2005
Citation: Oba, M., Baldwin, R.L., Owens, S.L., Bequette, B.J. 2005. Metabolic fates of ammonia nitrogen in ruminal epithelial and duodenal mucosal cells isolated from growing sheep. Journal of Dairy Science. 88:3963-3970.

Interpretive Summary: Energetically speaking the visceral tissues are costly to the productive efficiency of the ruminant animals. Because nitrogen metabolism by ruminant gut is potentially a site to improve efficiency of nutrient use, this study was conducted to assess if nitrogen from ammonia could be detoxified by sheep gut tissues. Ruminal epithelial cells (REC; cells that line the rumen) and duodenal mucosal cells (DMC; cells that line the small intestine) were isolated from growing sheep that had been fed a well balanced pelleted diet. Cells were incubated for 1 h with glucose, glutamate, ammonium chloride labeled with a special form of nitrogen which is detectably different from normal nitrogen (four concentrations 0, 5, 10, 20, or 40 mM), and one of four combinations of substrates to support urea synthesis. Incorporation of labeled ammonia into alanine, citrulline, arginine, and urea was determined by gas chromatography-mass spectrometry. For both cell types, ammonia-N transfer to alanine was lower when media contained N-carbamoyl-glutamate (a stable compound that mimics a known intermediate needed for urea synthesis) compared with control, whereas use of ammonia-N for alanine synthesis increased with ammonia concentration regardless of treatment. For REC, ammonia-N was not incorporated into citrulline, arginine or urea, and it was not incorporated into arginine or urea by DMC. Ammonia-N use for citrulline synthesis decreased media ammonia concentration increased. Alanine synthesis may be a significant metabolic pathway for ruminant gut tissues to detoxify ammonia when it is present at high concentrations in the rumen as compared to detoxification by the ornithine-urea cycle. Furthermore, DMC do exhibit a metabolic capability to incorporate ammonia-N into citrulline, but low or lack of activity of downstream enzymes of the ornithine-urea cycle appear to limit ammonia-N transfers to urea. Manipulation of ammonia N metabolism by the gut tissues is another promising nutritional or physiological approach to reduce net flux of ammonia and enhance net efficiency of N utilization. A better understanding of these processes will lead to formulation of rations and development of management strategies to increase efficiency of production.

Technical Abstract: The objective of this experiment was to determine the capability of ruminant gut tissues to detoxify ammonia-N using short-term incubations of isolated cells in vitro. Ruminal epithelial cells (REC) and duodenal mucosal cells (DMC) were isolated from growing Texel-Polypay ram lambs (n=4) fed a pelleted forage:concentrate based diet. Immediately after isolation, primary cells were incubated for 60 min with glucose (1mM), glutamate (1mM), [15N]ammonium chloride (0, 5, 10, 20, or 40 mM), and one of four combinations of substrates (1 mM each) that could support urea synthesis (control, N-carbamoylglutamate (NCG); NCG + ornithine (ONCG); ONCG + aspartate (AONCG)). Treatments were arranged in a 5 x 4 factorial design. Incorporation of ammonia-15N into alanine, citrulline, arginine, and urea was determined by gas chromatography-mass spectrometry. For both cell types, ammonia-N transfer to alanine was lower when incubation media contained NCG compared with control, whereas utilization of ammonia-N for net alanine synthesis increased (P< 0.001; quadratic) with ammonia concentration regardless of substrate treatment. For REC, ammonia-N was not incorporated into citrulline, arginine or urea, and neither was it incorporated into arginine or urea by DMC. Ammonia-N utilization for net citrulline synthesis exhibited an inverse relationship with ammonia concentration, decreasing linearly (P< 0.001) as media ammonia concentration increased. Thus, alanine synthesis may be a significant metabolic pathway for ruminant gut tissues to detoxify ammonia-N when it is presented luminally at high concentrations as compared to detoxification by the ornithine-urea cycle. Furthermore, DMC do exhibit a metabolic capability to incorporate ammonia-N into citrulline, but low or lack of activity of downstream enzymes of the ornithine-urea cycle appears to limit ammonia-N transfers to urea.