Hostname: page-component-cd9895bd7-8ctnn Total loading time: 0 Render date: 2024-12-28T00:48:48.044Z Has data issue: false hasContentIssue false
  • English
  • Français

Use of stable isotopes to measure de novo synthesis and turnover of amino acid-C and -N in mixed micro-organisms from the sheep rumen in vitro

Published online by Cambridge University Press:  09 March 2007

C. Atasoglu  and
A. Y. Guliye
C. Atasoglu
Affiliation:
Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, UK
A. Y. Guliye*
Affiliation:
Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, UK
*
*Corresponding author: Dr R. J. Wallace, fax +44 1224 716687, email rjw@rri.sari.ac.uk
Rights & Permissions [Opens in a new window]

Abstract

Core share and HTML view are not available for this content. However, as you have access to this content, a full PDF is available via the ‘Save PDF’ action button.

Protein synthesis and turnover in ruminal micro-organisms were assessed by stable-isotope methods in order to follow independently the fate of amino acid (AA)-C and -N in different AA. Rumen fluid taken from sheep receiving a grass hay–concentrate diet were strained and incubated in vitro with starch–cellobiose–xylose in the presence of NH3 and 5 g algal protein hydrolysate (APH)/l, in incubations where the labels were 15NH3, [15N]APH or [13C]APH. Total 15N incorporation was calculated from separate incubations with 15NH3 and [15N]APH, and net N synthesis from the increase in AA in protein-bound material. The large difference between total and net AA synthesis indicated that substantial turnover of microbial protein occurred, averaging 3·5 %/h. Soluble AA-N was incorporated on average more extensively than soluble AA-C (70 v. 50 % respectively, P=0·001); however, incorporation of individual AA varied. Ninety percent of phenylalanine-C was derived from the C-skeleton of soluble AA, whereas the incorporation of phenylalanine-N was 72 %. In contrast, only 15 % aspartate-C + asparagine-C was incorporated, while 45 % aspartate-N+asparagine-N was incorporated. Deconvolution analysis of mass spectra indicated substantial exchange of carboxyl groups in several AA before incorporation and a condensation of unidentified C2 and C4 intermediates during isoleucine metabolism. The present results demonstrate that differential labelling with stable isotopes is a way in which fluxes of AA synthesis and degradation, their biosynthetic routes, and separate fates of AA-C and -N can be determined in a mixed microbial population.

Keywords

Amino acidsCarbon skeletonsRumenSheep
Type
Research Article
Information
British Journal of Nutrition , Volume 91 , Issue 2 , February 2004 , pp. 253 - 261
Copyright
Copyright © The Nutrition Society 2004

References

Allison, MJ (1969) Biosynthesis of amino acids by ruminal microorganisms. J Anim Sci 29, 797–807.CrossRefGoogle ScholarPubMed
Allison, MJ (1965) Phenylalanine biosynthesis from phenylacetic acid by anaerobic bacteria from the rumen. Biochem Biophys Res Commun 18, 30–35.CrossRefGoogle ScholarPubMed
Amin, MR & Onodera, R (1997) Synthesis of phenylalanine and production of other related compounds from phenylpyruvic acid and phenylacetic acid by ruminal bacteria, protozoa, and their mixture in vitro J Gen Appl Microbiol 43, 9–15.CrossRefGoogle ScholarPubMed
Argyle, JL & Baldwin, RL (1989) Effects of amino acids and peptides on rumen microbial growth yields. J Dairy Sci 72, 2017–2027.CrossRefGoogle ScholarPubMed
Armstead, IP & Ling, JR (1993) Variations in the uptake and metabolism of peptides and amino acids by mixed ruminal bacteria in vitro. Appl Environ Microbiol 59, 3360–3366.CrossRefGoogle ScholarPubMed
Atasoglu, CValdés, CNewbold, CJ & Wallace, RJ (1999) Influence of peptides and amino acids on fermentation rate and de novo synthesis of amino acids by mixed micro-organisms from the sheep rumen. Br J Nutr 81, 307–314.CrossRefGoogle ScholarPubMed
Atasoglu, C, Valdés, C, Walker, ND, Newbold, CJ & Wallace, RJ (1998) De novo synthesis of amino acids by the ruminal bacteria Prevotella bryantii B1 4, Selenomonas ruminantium HD4, and Streptococcus bovis ES1. Appl Environ Microbiol 64, 2836–2843.CrossRefGoogle Scholar
Blake, JS, Salter, DN & Smith, RH (1983) Incorporation of nitrogen into rumen bacterial fractions of steers given protein- and urea-containing diets. Ammonia assimilation into intracellular bacterial amino acids. Br Jn Nutr 50, 769–782.CrossRefGoogle ScholarPubMed
Broderick, GA & Balthrop, JE (1979) Chemical inhibition of amino acid deamination by ruminal microbes in vitro. J Anim Sci 49, 1101–1111.CrossRefGoogle Scholar
Calder, AG & Smith, A (1988) Stable isotope ratio analysis of leucine and isocaproic acid in blood plasma by gas chromatography/mass spectrometry Rapid Commun Mass Spectrom 2, 14–16.CrossRefGoogle Scholar
Campbell, IM (1974) Incorporation and dilution values – their calculation in mass spectrally stable isotope labeling experiments Bioorg Chem 3, 386–397.CrossRefGoogle Scholar
Carro, MD & Miller, EL (1999) Effect of supplementing a fibre basal diet with different nitrogen forms on ruminal fermentation and microbial growth in an in vitro semi-continuous culture system (RUSITEC). Br J Nutr 82, 149–157.CrossRefGoogle Scholar
Chalupa, W (1976) Degradation of amino acids by the mixed rumen microbial population J Anim Sci 43, 828–834.CrossRefGoogle ScholarPubMed
Cotta, MA & Russell, JB (1982) Effect of peptides and amino acids on efficiency of rumen bacterial protein synthesis in continuous culture. J Dairy Sci 65, 226–234.CrossRefGoogle Scholar
Cruz Soto, R, Muhammed, SA, Newbold, CJ, Stewart, CS & Wallace, RJ (1994) Influence of peptides, amino acids and urea on microbial activity in the rumen of sheep receiving grass hay and on the growth of rumen bacteria in vitro. Anim Feed Sci Technol 49, 151–161.CrossRefGoogle Scholar
Demeyer, DI & Van Nevel, CJ (1979) Effect of defaunation on the metabolism of rumen micro-organism. Br j Nutr 42, 515–524.CrossRefGoogle Scholar
Gorosito, AR, Russell, JB & Van Soest, PJ (1985) Effect of carbon-4 or carbon-5 volatile fatty acids on digestion of plant cell wall in vitro. J Dairy Sci 68, 840–847.CrossRefGoogle Scholar
Hungate, RE & Stack, RJ (1982) Phenylpropanoic acid: growth factor for Ruminococcus albus. Appl Environ Microbiol, 44, 79–83.CrossRefGoogle ScholarPubMed
McDonald, P, Edwards, RA & Greenhalgh, JFD (1995) Animal Nutrition New York: Longman Scientific and Technical.Google Scholar
Maeng, WJ, Van Nevel, CJ, Baldwin, RL & Morris, JG (1976) Rumen microbial growth rates and yields: effects of amino acids and proteins. J Dairy Sci 59, 68–79.Google Scholar
Masson, HA & Ling, JR (1986) The in vitro metabolism of free and bacterially-bound 2,2'-diaminopimelic acid by rumen microorganisms. J Appl Bacteriol 60, 341–349.CrossRefGoogle Scholar
Menke, KH & Steingass, H (1988) Estimation of the energetic feed value obtained from chemical analysis and in vitro gas production using rumen fluid. Anim Res Dev 28, 8–55.Google Scholar
Mir, PS, Mir, Z & Robertson, JA (1986) Effect of branched-chain amino acids or fatty acid supplementation on in vitro digestibility of barley straw or alfalfa hay Can J Anim Sci 66, 151–156.CrossRefGoogle Scholar
Nieto, R, Calder, AG, Anderson, SE & Lobley, GE (1996) Method for the determination of 15NH3 enrichment in biological samples by gas chromatography/electron impact mass spectrometry. J Mass Spectrom 31, 289–294.3.0.CO;2-Z>CrossRefGoogle Scholar
Nolan, JV (1975) Quantitative models of nitrogen metabolism in sheep. In Digestion and Metabolism in the Ruminant, pp. 416–431 [McDonald, IW and Warner, ACI, editors]. Armidale, New South Wales: University of New England Publishing Unit.Google Scholar
Nolan, JV, Norton, BW & Leng, RA (1976) Further studies on the dynamics of nitrogen metabolism in sheep. Br J Nutr 35, 127–147.CrossRefGoogle ScholarPubMed
Nolan, JV & Stachiw, S (1979) Fermentation and nitrogen dynamics in merino sheep given a low-quality-roughage diet. Br J Nutr 42, 63–80.CrossRefGoogle Scholar
Oltjen, RR, Slyter, LL, Williams, EE & Kern, DL (1971) Influence of the branched-chain volatile fatty acids and phenylacetate on ruminal microorganisms and nitrogen utilization by steers fed urea and isolated soyprotein. J Nutr 101, 101–112.CrossRefGoogle Scholar
Onodera, R & Kandatsu, M (1973) Synthesis of lysine from a, e-diaminopimelic acid by mixed ciliated rumen protozoa. Nat New Biol 244, 31–32.CrossRefGoogle Scholar
Onodera, R, Takashima, H & Ling, JR (1991) In vitro production of lysine from 2,2'-diaminopimelic acid by rumen protozoa. J Protozool 38, 421–425.CrossRefGoogle Scholar
Purser, DB & Buechler, SM (1966) Amino acid composition of rumen organisms. J Dairy Sci 49, 81–84.Google Scholar
Robinson, IM & Allison, MJ (1969) Isoleucine biosynthesis from 2-methylbutyric acid by anaerobic bacteria from the rumen. J Bacteriol 97, 1220–1226.CrossRefGoogle Scholar
Russell, JB (1983) Fermentation of peptides by Bacteroides ruminicola B14. Appl Environ Microbiol 45, 1566–1574.CrossRefGoogle ScholarPubMed
Russell, JB & Sniffen, CJ (1984) Effect of carbon-4 and carbon-5 volatile fatty acids on growth of mixed rumen bacteria in vitro. J Dairy Sci 67, 987–994.CrossRefGoogle ScholarPubMed
Sauer, FD, Erfle, JD & Mahadevan, S (1975) Amino acid biosynthesis in mixed rumen cultures. Biochem J 150, 357–372.CrossRefGoogle ScholarPubMed
Shimbayashi, KObara, Y & Yonemura, T (1975) Changes of free amino acids during rumen fermentation and incorporation of urea-15 N into microorganisms in vitro. Jpn J Zootech Sci 46, 243–250.Google Scholar
Stack, RJ, Hungate, RE & Opsahl, WP (1983) Phenylacetic acid stimulation of cellulose digestion by Ruminococcus albus 8. Appl Environ Microbiol 46, 539–544.CrossRefGoogle ScholarPubMed
Storm, E & Ørskov, ER (1983) The nutritive value of rumen micro-organisms in ruminants 1. Large-scale isolation and chemical composition of rumen micro-organisms, Br J Nutr 50, 463–470.CrossRefGoogle ScholarPubMed
Van Nevel, CJ & Demeyer, DI, (1977) Determination of rumen microbial growth in vitro from 32P-labelled phosphate incorporation. Br J Nutr 38, 101–114.CrossRefGoogle ScholarPubMed
Virtanen, AI (1966) Milk production of cows on protein-free feed. Science 153, 1603–1614.CrossRefGoogle ScholarPubMed
Wallace, RJ (1994) Amino acid and protein synthesis, turnover, and breakdown by ruminal microorganisms In Principles of Protein Nutrition of Ruminants, pp. 71–111 [Asplund, jmeditor]. Boca Raton, FL: CRC Press, Inc.Google Scholar
Wallace, RJ (1979) Effect of ammonia concentration on the composition, hydrolytic activity and nitrogen metabolism of the microbial flora of the rumen. J Appl Bacteriol 47, 443–455.CrossRefGoogle ScholarPubMed
Wallace, RJ & McPherson, CA (1987) Factors affecting the rate of breakdown of bacterial protein in rumen fluid Br J Nutr 58, 313–323.CrossRefGoogle ScholarPubMed