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Program of Cellular and Molecular Biosciences, Department of Animal Sciences, Auburn University, Auburn, AL 36849-5415
1To whom correspondence should be addressed. E-mail: bergewg{at}auburn.edu.
See related article: J. Nutr. 134: 3378–3384, 2004.
The development of dietary strategies to enhance the efficiency of nitrogen utilization by ruminants during agricultural production necessitates experimental and analytical methods that can track the multiple nitrogenous constituents through the complex digestive tract of these animals. In particular, past methods for tracking the passage of ruminal ciliate protozoa to the small intestine and assessing the contribution of protozoal protein to the animal’s protein nutriture have been unsatisfactory. Sylvester et al. (1) have now developed and validated a quantitative real-time PCR-based assay system to specifically track rumen protozoa that can be applied to animal studies in vivo.
A unique anatomic and physiologic symbiotic arrangement in ruminant animals allows well-adapted anaerobic microorganisms (bacteria, ciliate protozoa, and fungi) to colonize the forestomachs (reticulo-rumen) of the host’s gastrointestinal tract and animals to utilize end-products arising from microbial activity in the reticulo-rumen. Dietary components consumed by ruminants first undergo partial to total dissimilation by the rumen-reticulum anaerobic fermentation. Ingested carbohydrates are hydrolyzed and fermented to volatile fatty acids (VFA),2 whereas proteins are degraded to peptides, amino acids, and ultimately ammonia. Rumen microorganisms utilize metabolic energy derived from carbohydrate fermentation and amino acids and/or ammonia arising from degradation of proteins and/or nonprotein nitrogen (NPN; principally urea) as nitrogen sources for ruminal microbial protein synthesis. End-products of the rumen fermentation are constantly removed, which enables the fermentation to be continually operative (2). The microbiology of the reticulo-rumen ecosystem was studied extensively, and rumen contents contain 
1010 bacterial and 105–106 protozoal cells/mL. Although bacterial concentrations tend to rise and fall with feeding patterns, substrate availability, and feed fermentation, protozoal growth dynamics are more complex (2,3). These anaerobic, eukaryotic organisms feed on bacteria, utilize sugars arising in part from bacterial metabolism, may rapidly synthesize and store excess starch after feeding, and may undergo autolysis. Despite their prevalence in the reticulo-rumen, protozoa appear to account for no more than 20–30% of total microbial biomass flow to the abomasum (4).
End-products arising from ruminal microbial activity are microbial biomass, VFA, and partially degraded feed components. Although VFA are absorbed directly across the rumen epithelium into the blood supply, the remaining end-products pass out of the rumen via the omasal orifice to the abomasum (gastric stomach) and then to the small intestine. Workers in the early 20th century already appreciated that the microbial biomass was the principal supply of amino acids for the animal, whereas VFA were the primary energy supply of ruminants (2). Subsequent work showed that microbial protein contained all of the indispensable amino acids even in animals fed only NPN and is of medium to high nutritive quality (2,5).
To improve productivity, the digestive physiology and nutrition of ruminants have been studied intensely for more than 100 y. Despite the complex anatomical and physiologic properties of the ruminant digestive tract, researchers in the first half of the 20th century were able to describe qualitative and quantitative aspects of energy metabolism in these animals (2). When it came to assessing dietary protein adequacy and amino acid needs of ruminants, however, their digestive physiology made it difficult to measure with precision total amino acids passing to the small intestine and to determine amino acid requirements for maintenance, growth, and lactation. From the perspective of the amino acid supply, it became clear that dietary protein intake does not quantitatively reflect actual proteins/amino acids passage [composed of bacterial, protozoal and fungal biomass and undegraded dietary protein (UDP)] to the abomasum. In addition, from the perspective of amino acid requirements, amino acid needs could not be determined by empirical approaches such as feeding different amounts of dietary proteins or amino acids and measuring production responses because dietary nitrogen sources are degraded by rumen microorganisms. Thus, quantitative assessment of all aspects of protein nutrition in ruminants has been and continues to be a major challenge (4).
Workers in the field have long appreciated that quantitation of rumen microbial biomass production and UDP passage requires measures of digesta flow and identification of bacterial, protozoal, and fungal biomass as well as UDP fractions in the digesta (6,7). Early workers tried to estimate rumen microbial biomass production by emptying total rumen contents via a rumen fistula to determine acid precipitable N and by measuring rumen content turnover in vivo with nondigestible markers. Here, total rumen protein mass times daily rumen turnover rate provided an estimate of total daily protein flow to the small intestine and reticulo-rumen microbial biomass production when animals were fed only NPN sources (2).
In essence, all contemporary procedures to quantify rumen output of microbial protein and UDP measure both rate of digesta passage to either the omasum, abomasum or duodenum and the concentration of the various protein fractions in intestinal contents (5,6,7). Because digesta flow is influenced by feed intake patterns and rumen turnover dynamics, to minimize the influence of feeding patterns and the quasi-continuous nature of rumen outflow on recovery of total microbial biomass flow, sampling protocols spread over several days to obtain equally spaced samples for a representative 24-h period were adopted. Individual samples were then pooled and analyzed for a digesta flow marker (rare earth or nondigestible feed component), total N and markers for microbial fractions (6,7).
Although total protein passage can be determined by total N analysis, quantifying bacterial and protozoal protein requires the use of specific markers for each microbial fraction. Over the last 50 y, workers have used radiolabeled and heavy isotope incorporation, protozoal- (2-amino ethylphosphonic acid; AEP) and bacterial- (2,6 diaminopimelic acid; D-alanine) specific cell constituents, or nucleic acids (RNA; DNA) as markers for bacterial and protozoal biomass production. Isotope incorporation approaches suffer from isotope equilibration and recycling, and difficulties in determining specific activities of precursor pools. Neither organism-specific markers such as AEP, which was first thought to be present only in ruminal protozoa but has also been found in ruminal bacterial biomass preparations, nor nucleic acids in themselves could distinguish between bacterial and protozoal biomass (4). Recent advances in the identification of microorganisms using PCR technology enabled workers to identify specific organisms in complex ecosystems utilizing small ribosomal subunit rDNA (16S rDNA for prokaryotic and 18S rDNA for eukaryotic organisms, respectively) as markers (8). These approaches were applied specifically to rumen protozoal biomass by Sylvester et al. (1). In a comprehensive fashion, the authors detailed the complete assay procedures including sampling of rumen contents; isolation of ciliate protozoa; sampling of duodenal digesta; extraction and purification of DNA; real-time PCR (including primer sequences) for measurement of DNA encoding eukaryotic small-subunit RNAs; purification of ruminal protozoa to determine representative 18S rDNA marker-to-protein ratios and duodenal protozoal-specific 18S rDNA copy numbers. For every step of this assay, the efficiency of adopted procedures, the extent of sampling necessary and sample storage, recovery experiments, utility of purification protocols, and real-time PCR protocols were exhaustively validated. Although adoption of this comprehensive assay will require laboratory skills and physical resources associated with contemporary molecular biology, this new approach will provide new insight into the contribution of rumen protozoa to protein metabolism in ruminants hitherto considered impossible. In time, this new knowledge will result in improved diet formulations and enhanced efficiency of N utilization by ruminants.
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FOOTNOTES |
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Manuscript received 8 September 2004. Revision accepted 12 September 2004.
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LITERATURE CITED |
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 TOP LITERATURE CITED |
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1. Sylvester, J. T., Karnati, S.K.R., Zhongtang, Y., Morrison, M. & Firkins, J. L. (2004) Development of an assay to quantify rumen ciliate protozoal biomass in cows using real-time PCR. J. Nutr. 134:3378-3384.
2. Hungate, R. E. (1966) The Rumen and Its Microbes 1966 Academic Press New York, NY.
3. Dehority, B. A. (2003) Rumen Microbiology 2003 Nottingham University Press Nottingham, UK.
4. Steinhour, W. D. & Clark, J. H. (1982) Microbial protein flow to the small intestine of ruminants. Owens, F. N. eds. Protein Requirements for Cattle 1982:166-182 MP-109, Oklahoma State University, Division of Agriculture Stillwater, OK. .
5. Owens, F. N. & Bergen, W. G. (1983) Nitrogen metabolism of ruminant animals: historical perspective, current understanding and future implications. J. Anim. Sci. 57(suppl. 2):498-518.
6. Boggs, D. L., Bergen, W. G. & Hawkins, D. R. (1987) Effects of tallow supplementation and protein withdrawal on ruminal fermentation, microbial synthesis and site of digestion. J. Anim. Sci. 64:907-914.
7. Hussein, H. S., Merchen, N. R. & Fahey, G. C., Jr (1996) Effects of forage percentage and canola seed on ruminal protein metabolism and duodenal flows of amino acids in steers. J. Dairy Sci. 79:98-104.[Abstract]
8. Gailbraith, E. A., Antonopoulos, D. A. & White, B. A. (2004) Suppressive subtractive hybridization as a tool for identifying genetic diversity in an environmental metagenome: the rumen as a model. Environ. Microbiol. 6:928-937.[Medline]
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  J. T. Sylvester, S. K. R. Karnati, Z. Yu, M. Morrison, and J. L. Firkins Development of an Assay to Quantify Rumen Ciliate Protozoal Biomass in Cows Using Real-Time PCR J. Nutr., December 1, 2004; 134(12): 3378 - 3384. [Abstract] [Full Text] [PDF]
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