Refinements in primary rumen epithelial cell culture techniques

The objectives of this study were to 1) determine if number of rumen epithelial cells incubated in a primary cell culture affects the rate of metabolite production; and 2) determine the optimum mode of data expression to standardize reporting criteria. A section of rumen epithelial tissue was excised from five Holstein heifers and subjected to serial tryptic digestion to isolate cells. Isolated cells had a mean viability of 85.8% (±1.29) and were incubated at concentrations of .5-, 1-, 5-, 10-, 20-, and 40-million cells per flask. The oxidation of [l-¹⁴C]butyrate to ¹⁴CO₂ and production of acetoacetate (ACAC), β-hydroxybutyrate (βHBA), lactate, and pyruvate were measured for cell dilution comparisons. Cell dry matter, cell total protein, epithelial wet tissue weight, body weight, and metabolic body weight were measured to generate twelve different forms of data expression. Coefficients of variation were calculated for each type of expression. Expressing data per cell number resulted in the lowest variation (P < .01). The oxidation of [l-¹⁴C]butyrate to ¹⁴CO₂ did not significantly differ between cell dilutions after 90-minute incubations. βHBA and lactate concentrations were the largest at the 1-million cell dilution, (P < .05 and P < .01). ACAC and pyruvate had largest concentrations (P < .05 and P < .10) produced at .5-million cells/flask. The .5- and 1- million cell dilutions had low mitochondrial redox potentials with respective βHBA to ACAC ratios of 1.04 and 1.14 indicating aberrant metabolism. Production of ACAC and pyruvate did not differ between 5-, 10-, 20-, or 40-million cell concentrations. Conversely, βHBA concentrations significantly decreased (P < .001) as cell concentrations increased from 5- to 40-niillion. Similarly, lactate concentrations appeared to decrease (P = .05) as cell concentrations increased from 5- to 40-million. The suggested range of rumen epithelial cells to include in incubations is 5- to 10- million cells/flask. This will minimize large variation caused by using low cell numbers and potential for reduced metabolite production caused by incubating large cell quantities. When rumen tissue taken from animals of the same species, size, and stage of development; data expressed on a per cell number basis is preferred. However, it is recommended that cell protein, cell dry matter, and animal metabolic weight also be included for future comparisons between species and laboratories.