Evaluation of primary rumen epithelial cell incubation 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 establish an ideal mode of data expression in order to standardize the reporting criteria for primary cell incubation. Epithelial tissue was excised from five Suffolk×Dorset cross-bred sheep and subjected to serial tryptic digestion to isolate cells. Isolated cells were incubated for 90 min in 25 mM propionate and 10 mM butyrate at concentrations of 0.5, 1, 5, 10, 20 and 40 million cells per flask (total volume=3 ml). Production of acetoacetate (ACAC), β-hydroxybutyrate (BHBA), lactate (LAC) and pyruvate (PYR) were measured. Data were expressed as either cell number, cell dry matter (DM) or cell crude protein (CP) alone or per epithelial wet tissue weight, body weight (BW) or metabolic BW to generate 12 different forms of data expression. Coefficients of variation (CVs) were calculated for all 12 modes of expression. Expressing data per cell number resulted in the lowest variation (P<0.01) and data adjusted for metabolic BW had less variation than BW. ACAC concentrations were largest at 0.5 million cells per flask (P<0.05) and there were no differences between 1, 5, 10 and 20, and only 40 differed from 0.5 and 5 million cells per flask. Concentrations of BHBA were largest at 1 and 5 million cells per flask, but were different (P<0.05) only from 20 and 40 million cells per flask. LAC and PYR concentrations were largest at 1 million cells per flask, but no significant differences were found. Ratios of BHBA:ACAC were below one for the 0.5 million cells per flask indicating low mitochondrial redox potentials (P<0.05). A suggested range of rumen epithelial cells to include in incubations is 5-20 million cells per 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 CP and cell DM be included to facilitate future comparison between laboratories and species.