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Metabolic Fates of Ammonia-N in Ruminal Epithelial and Duodenal Mucosal Cells Isolated from Growing Sheep

¹ Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Canada T6G 2P5
² Bovine Functional Genomics Laboratory, Animal and Natural Resources Institute, USDA-ARS, Beltsville, MD 20705
³ Department of Animal and Avian Sciences, University of Maryland, College Park 20742

Corresponding author: M. Oba; e-mail: masahito.oba{at}ualberta.ca.

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 (5, 10, 20, or 40 mM), and 1 of 4 combinations of substrates (1 mM each) that could support urea synthesis [control, N-carbamoylglutamate (NCG); NCG + ornithine (ONCG); and ONCG + aspartate (AONCG)]. Treatments were arranged in a 4 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 medium contained NCG compared with control, whereas use of ammonia-N for net alanine synthesis increased quadratically with ammonia concentration regardless of substrate treatment. For REC, ammonia-N was not incorporated into citrulline, arginine, or urea, nor into arginine or urea by DMC. Ammonia-N use for net citrulline synthesis exhibited an inverse relationship with ammonia concentration, decreasing linearly as media ammonia concentration increased. Thus, ala-nine 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 with detoxification by the ornithine-urea cycle. Furthermore, DMC do exhibit a metabolic capability to incorporate ammonia-N into citrulline, but low or absent activity of downstream enzymes of the ornithine-urea cycle appears to limit ammonia-N transfers to urea.

Key Words: sheep • ruminal epithelial cells • duodenal mucosal cells • ammonia

Abbreviation key: AONCG = ONCG + aspartate, DMC = duodenal mucosal cells, GC-MS = gas chromatography/mass spectrometry, NCG = N-carbamoylglutamate, ONCG = NCG + ornithine, REC = ruminal epithelial cells, t-BDMS = t-butyldimethylsilyl.

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