Evaluation of primary rumen epithelial cell culture techniques in sheep

Objectives of this study were to determine if the number of cells incubated in primary rumen epithelial cell cultures affects production rates of metabolites and to standardize reporting criteria by obtaining an optimum mode of data expression. Epithelial tissue was excised from five Suffolk x Dorset crossbred sheep and subjected to serial tryptic digestion to isolate cells. Isolated cells were incubated for 90-minutes in 25 mM propionate and 10 mM butyrate at concentrations of 0.5, 1, 5,10, 20 and 40-million cells per flask (total vol. = 3mL). Production of acetoacetate (AcAc), β-hydroxybutyrate (β-HBA), lactate (LAC) and pyruvate (PYR) were measured. Data were expressed as either cell number, cell dry matter or cell total protein alone or per epithelial wet tissue weight, body weight (BW) or metabolic BW to generate twelve different forms of data expression. Coefficients of variation were calculated for all 12 modes of expression. Expressing data per cell number resulted in the lowest variation (P < .01) and data adjusted for metabolic BW had less variation than BW. Acetoacetate concentrations were largest at 0.5-million cells/flask (P < .05) and there were no differences between 1, 5,10 and 20 and only 40 differed from 0.5 and 5-million cells/flask. β-HBA concentrations were largest at 1 and 5-million cells/flask. However, 1 and 5 only differed significantly (P < .05) from 20 and 40-million cells/flask. Lactate and PYR concentrations were largest at 1-million cells/flask, but no significant differences were found. Ratios of β-HBA:AcAc were below one for the 0.5-million cells/flask indicating low mitochondrial redox potentials (P < .05). A suggested range of rumen epithelial cells to include in incubations is 5 to 20-million/flask. This range will minimize the potential for altered metabolite production caused by incubating large cell quantities as well as the experimental error associated with using low cell numbers. When rumen tissue is taken from animals of the same species, size and stage of development, data adjusted by cell number is preferred. However, it is recommended that metabolic BW, cell protein and cell dry matter be included to facilitate future comparison between laboratories and species.