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United States Department of Agriculture

Agricultural Research Service

Title: Refinements in Primary Rumen Epithelial Cell Culture Techniques

Authors
item Klotz, J - UNIVERSITY OF TENNESSEE
item BALDWIN, RANSOM
item Gillis, R - UNIVERSITY OF TENNESSEE
item Heitmann, R - UNIVERSITY OF TENNESSEE

Submitted to: Journal of Dairy Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 20, 2000
Publication Date: N/A

Interpretive Summary: The objectives of this research were to determine if number of rumen epithelial cells incubated in a primary cell culture affects the rate of production of important metabolites for ruminant animals and to determine the best way to report the findings. We isolated cells from the largest compartment of the ruminant stomach and evaluated the way that the cells used nutrients. We found that expressing data on a per cell basis was the best. We ultimately determined that the range of rumen epithelial cells to include in incubations is 5- to 20-million cells/flask which will minimize errors associated with using low cell numbers and the potential for reduced metabolite production caused by incubating large numbers of cells. When rumen tissue taken from animals of the same species, size, and stage of development; data adjusted by cell number is preferred. However, it is recommended that cell protein, cell dry matter, and animal metabolic weight be included to facilitate future comparison between species and laboratories.

Technical Abstract: The objectives were to determine if number of rumen epithelial cells incubated in a primary cell culture affects the rate of metabolite production and determine the optimum mode of data expression to standardize reporting criteria. Isolated rumen epithelial cells had a mean viability of 85.8% (± 1.29) and were incubated at concentrations of 0.5-, 1-, 5-, 10-, 20-, and 40-million cells per flask. The oxidation of butyrate to CO2 and production of acetoacetate (ACAC), b-hydroxybutyrate (bHBA), lactate, and pyruvate were evaluated. Cell number, dry matter, protein, epithelial wet weight, body weight, and metabolic body weight were measured to generate twelve forms of expression and coefficients of variation were calculated. Expressing data per cell number resulted in the lowest variation. Oxidation of butyrate to 14CO2 and pyruvate production did not differ between cell dilutions. Lactate and ACAC concentrations were greatest at 0.5- and 1-million cells/flask, respectively, with no differences between 5- to 40-million cells/flask. Production of bHBA was greater for 1- than 0.5- and 40-million cells/flask, but did not change between cell concentrations 5- to 20- million. Thus, the suggested range of rumen epithelial cells to include in incubations is 5- to 20-million cells/flask to minimize experimental error associated with using low cell numbers and potential for reduced metabolite production caused by incubating large cell quantities. For within species, size, and stage of development comparisons, data adjusted by cell number is preferred. However, it is recommended that cell protein, cell dry matter, and animal metabolic weight be included to facilitate future comparison between species and laboratories.

Last Modified: 9/10/2014
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